Utilization of cd39 and cd103 for identification of human tumor reactive t cells for treatment of cancer

ABSTRACT

Methods are disclosed for treating a subject with a tumor. These methods include administering to the subject a therapeutically effective amount of CD8+CD39+CD103+ T cells. Methods also are disclosed for isolating a nucleic acid encoding a T cell receptor (TCR) that specifically binds a tumor cell antigen. These methods include isolating CD8+CD39+CD103+ T cells from a sample from a subject with a tumor expressing the tumor cell antigen, and cloning a nucleic acid molecule encoding a TCR from the CD8+CD39+CD103+ T cells. In addition, methods are disclosed for expanding CD8+CD39+CD103+ T cells. In additional embodiments, methods are disclosed for determining if a subject with a tumor will respond to a checkpoint inhibitor. The methods include detecting the presence of CD8+CD39+CD103+ T cells in a biological sample from a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 16/616,932,filed on Nov. 25, 2019, which is the U.S. National Stage ofInternational Application No. PCT/US2018/031197, filed May 4, 2018,which claims the benefit of U.S. Provisional Application No. 62/517,612,filed Jun. 9, 2017. The prior applications are all herein incorporatedby reference.

FIELD

This relates to the field of cancer, specifically to the use of adoptivetransfer of cells, such as CD8⁺CD39⁺CD103⁺ T cells, and the use of Tcell receptors (TCRs), such as from CD8⁺CD39⁺CD103⁺ T cells, for thetreatment of tumors, and related to methods for assessing treatment,such as by measuring CD8⁺CD39⁺CD103⁺ T cells.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The electronic sequence listing (6727-98940-11_Sequence_Listing.xml;Size: 29,152 bytes; and Date of Creation: Dec. 28, 2022) is hereinincorporated by reference in its entirety.

BACKGROUND

The immune system plays a major role in recognizing and killing tumorcells and new therapeutic approaches have focused on boosting and/orrestoring its function in cancer patients. An effective immune responseinvolves the concerted action of several different cell types amongwhich CD8 T cells are key players that can specifically recognize andkill cancer cells via the release of cytotoxic molecules and cytokines(Klebanoff C A et al., 211:214-24, Immunol. Rev., 2006).Tumor-infiltrating CD8 T cells recognize tumor-associated antigens(TAA's), which can comprise self-antigens that are overexpressed inneoplastic lesions, as well as tumor-specific antigens (TSA's) orneoantigens, which arise as a consequence of tumor-specific mutations(Finn O J, Cancer Immunol Res, 2017). According to the current paradigm,tumor-specific CD8 T cells are primed in tumor-draining lymph nodes andthen migrate via the blood to the tumor to exert their effectorfunction. Previous work has shown that tumor-infiltrating CD8 T cellsrepresent a heterogeneous population of cells comprised oftumor-specific T cells as well as bystander T cells with no tumorspecificity. The latter are recruited to the tumor site by theinflammation associated with tumor progression (proliferation,angiogenesis and metastasis). However, a need remains forimmunotherapeutic methods, wherein the efficacy/efficiency of adoptivetransfer is increased.

SUMMARY

The phenotype and function of tumor-reactive CD8 T cells was evaluated,and it was determined that co-expression of CD103 and CD39 identified apopulation of tumor-infiltrating CD8 T cells specifically induced in thetumor microenvironment. These cells, which are chronically stimulatedwithin the tumor, were highly enriched for tumor reactivity and had theability to kill autologous tumor cells. A higher frequency ofCD8⁺CD39⁺CD103⁺ T cells in human tumors correlated with a greateroverall survival. It was determined that the presence of CD8⁺CD39⁺CD103⁺T cells can indicate that a therapeutic method is effective for treatinga tumor. In addition, CD8⁺CD39⁺CD103⁺ T cells can be adoptivelytransferred into a subject to treat a tumor.

Methods are disclosed for treating a subject with a tumor, includingadministering to the subject a therapeutically effective amount ofCD8⁺CD39⁺CD103⁺ T cells, thereby treating the subject with the tumor. Insome embodiments, the method includes administering a therapeuticallyeffective amount of a Programmed Death (PD)-1 antagonist, a ProgrammedDeath Ligand (PD-L)1 antagonist, a Cytotoxic T-lymphocyte-AssociatedProtein 4 (CTLA-4) antagonist, a B- and T-lymphocyte Attenuator (BTLA)antagonist, T-cell Immunoglobulin and Mucin-domain containing-3 (TIM-3)antagonist, a Lymphocyte-Activation Gene 3 (LAG3) antagonist, or a 4-1BBagonist to the subject.

In some embodiments, methods are disclosed for isolating a nucleic acidsequence encoding a T cell receptor (TCR) that specifically binds atumor cell antigen. These methods include isolating CD8⁺CD39⁺CD103⁺ Tcells from a subject with a tumor expressing the tumor cell antigen, andcloning the nucleic acid sequence encoding a TCR molecule from theCD8⁺CD39⁺CD103⁺ T cells, thereby isolating a nucleic acid sequenceencoding the TCR specific for the tumor.

In additional embodiments, methods are disclosed for expandingCD8⁺CD39⁺CD103⁺ T cells. These methods include culturing CD8⁺CD39⁺CD103⁺T cells in a tissue culture medium comprising glutamine, serum, andantibiotics; stimulating the isolated T cells with an effective amountof allogenic irradiated feeder cells and a cytokine; and culturing thestimulated T cells in a tissue culture medium and an effective amount ofthe cytokine.

In yet other embodiments, methods are disclosed for determining if asubject with a tumor will respond to a cancer therapeutic, such ascheckpoint inhibitor, biological response modifier (for example,cytokines and chemokines), a cancer vaccine, chemotherapy and/orradiation. Methods are also disclosed for determining if a subject willrespond to a surgical procedure. The methods include detecting thepresence of CD8⁺CD39⁺CD103⁺ T cells in a biological sample from asubject, wherein the presence of the CD8⁺CD39⁺CD103⁺ T cells in thebiological sample indicates that the cancer therapeutic or the surgicalprocedure will be effective for treating the tumor in the subject. Insome non-limiting examples, the methods can also include administeringthe cancer therapeutic to the subject, or preforming the surgicalprocedure.

In further embodiments, methods are disclosed for determining if asubject with a tumor will respond to a cancer therapeutic. The methodsinclude administering to the subject a first dose of the cancertherapeutic, and determining the number of CD8⁺CD39⁺CD103⁺ T cells in abiological sample from the subject. An increase in the number ofCD8⁺CD39⁺CD103⁺ T cells in the biological sample as compared to acontrol indicates that the first dose of the cancer therapeutic iseffective for treating the tumor in the subject.

In more embodiments, methods are disclosed for treating a subject with atumor. The methods include administering to a subject a first dose ofthe cancer therapeutic, and determining the number of CD8⁺CD39⁺CD103⁺ Tcells in a biological sample from the subject. An increase in the amountof CD8⁺CD39⁺CD103⁺ T cells in the biological sample as compared to acontrol indicates that the first dose of the cancer therapeutic iseffective for treating the tumor in the subject. A second dose of thecancer therapeutic is administered to the subject, wherein the firstdose is the same as the second dose, or wherein the second dose is lowerthan the first dose.

In other embodiments, methods are disclosed for treating a subject witha tumor. The methods include administering to the subject a first doseof a cancer therapeutic, and determining the number of CD8⁺CD39⁺CD103⁺ Tcells in a biological sample from the subject. A decrease or no changein the amount of CD8⁺CD39⁺CD103⁺ T cells in the biological sample ascompared to a control indicates that the first dose of the cancertherapeutic is not effective for treating the tumor in the subject. Asecond dose of the cancer therapeutic is administered to the subjectwherein the second dose is higher than the first dose, or wherein thesecond dose is the same as the first dose.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F. Tumor-infiltrating CD103⁺CD8 T cells coexpress theectonucleotidase CD39. (A) Representative gating strategy for the flowcytometric analysis of T cells infiltrating a head and neck squamouscell carcinoma (HNSCC). Numbers in plots indicate the percent cells inrespective gates. (B) viSNE analysis of tumor infiltrating lymphocytes(TIL) gated on CD103+CD8 T cells as shown in (A). The gate identifiesCD103+CD8 T cells with the highest expression of CD39. The gate isapplied to all plots showing expression levels of PD-1, CD69 and CD127.(C) Flow cytometric analysis of CD8 TIL isolated from HNSCC, Melanoma,Ovarian and CRLM cancer patients. Numbers in each quadrant indicatepercent cells positive for CD39 and/or CD103 on CD3+CD8+ T cells. (D)Summary of the frequency of CD39+CD103+(DP) CD8 TIL among patients withdifferent solid malignancies. Shown are 40 HNSCC patients, 2 lung cancerpatients, 6 melanoma patients, 2 ovarian cancer patients, 3 rectalcancer patients, 3 patients with colon cancer and 6 patients with CRLM.The red circle highlights one colon patient with Lynch's syndrome,leading to microsatellite instability (MSI). (E) Flow cytometricanalysis of the percentage of DP CD8 T cells in peripheral blood, normalLN, primary tumor and metastatic LN from one HNSCC patient. Numbers ineach quadrant indicate percent cells positive for CD39 and/or CD103 onCD3+CD8+ T cells. Data are representative of 40 HNSCC patients analyzed(only 9 patients for normal LN and Met LN). Percentages are summarizedin (F).

FIGS. 2A-2D. Gene expression profiling of CD8+CD39+CD103+ TIL reveals aprofile reminiscent of T_(RM) cells. (A) Principal component analysis(PCA) of gene expression of sorted DN, SP and DP CD8 TIL isolated from 3HNSCC and 2 ovarian cancer patients. The analysis was performed on genesdifferentially expressed between double positive (DP) and doublenegative (DN) CD8 TIL (n=372). (B) Unsupervised clustering of samplesusing the 372 differentially expressed genes between DP and DN CD8 TIL.Dark grey color indicates downregulated genes, light grey indicatesupregulated genes. (C) Waterfall plot representing the fold change (log2) of the most upregulated and downregulated genes in DP vs DN subsets.Light grey identifies genes with a chronic activation signature; darkgrey indicates genes associated with the T_(RM) phenotype. (D)Expression of the top differentially expressed genes in each of the 5patients analyzed. The top row shows genes associated with the T_(RM)phenotype, bottom row indicates chronic activation signature genes. Eachsymbol represents a patient, which are connected with lines.

FIGS. 3A-3E. Phenotypic and functional properties of DP, SP and DN CD8TIL. (A) Ex vivo phenotypic analysis of the expression of surfaceproteins CD69, CCR7, CD127 and CD28, delineating a T_(RM) phenotype, onCD8 TIL (top). Summary of the frequency of above mentioned markers amongseveral cancer patients (n=6-13, bottom). *p<0.05; ***p<0.001;****p<0.0001 (ANOVA). (B) Ex vivo phenotypic analysis of the expressionof surface and intracellular proteins PD-1, CTLA-4, TIM-3, 4-1BB andKi-67, delineating an activated/chronically stimulated phenotype, on CD8TIL (top). Summary of the frequency of above mentioned markers amongseveral cancer patients (n=5-17, bottom). ***p<0.001; ****p<0.0001(ANOVA). (C) Representative flow cytometric analysis of IFN-γ, and TNF-αproduction by CD8 TIL subsets stimulated for 4 hrs with PMA/Ionomycin.Numbers in each quadrant indicate percent cells positive for IFN-γand/or TNF-α on CD3+CD8+ T cells. (D) Frequency of IFN-γ, TNF-α,IFN-γ/TNF-α double positive cells by CD8 TIL subsets. Data are from 6HNSCC patients. (E) Representative flow cytometric analysis of granzymeB production by CD8 TIL (left) and summary of 6 different HNSCC patients(right). *p<0.05; **p<0.01 (ANOVA).

FIG. 4 . Expression of CD39 and CD103 on CD8 T cells requires sustainedstimulation in a TGF-β rich milieu. Kinetics of CD39, CD103 and PD-1expression on sorted naive CD8 T cells from peripheral blood. Cells werestimulated with CD3/CD28 coated beads at a bead: T cell ratio of 1:2 inthe presence or absence of TGF-β (2 ng/ml) and expression of CD39, CD103and PD-1 was analyzed by flow cytometry at the indicated time points.Data are representative of 4 independent experiments.

FIGS. 5A-5D. DP CD8 TIL are highly clonal and share little overlap withany other CD8 T cell subsets. (A) Diversity of the TCRβ repertoirewithin the blood memory CD8 T cells, DN, SP and DP CD8 TIL. Thefrequencies of the most frequent clonotype, the 2nd to 5th mostfrequent, the 6th to 30th most frequent, and the rest of the clonotypesare shown for HPV+ and HPV− HNSCC patients and one ovarian cancerpatient. (B) The 500 most frequent CD8 TIL clonotypes are plotted basedon their frequency in DN, SP and DP CD8 TIL subsets. Each dot representsa distinct TCR clonotype. Dots on the axis indicate the clonotypesdetected within a single repertoire. (C) Same analysis as in (B)comparing the frequency of the top 500 clonotypes in memory CD8 T cellsin blood and normal LN to the frequency of the top 500 clonotypes in DNand DP CD8 TIL subsets. (D) TCRβ sequence overlap analyzed using theMorisita-Horn index between the DP CD8 TIL, and the blood memory CD8 Tcells, DN CD8 TIL and SP CD8 TIL. A TCRβ sequence overlap of 1 indicates100% similarity between two populations.

FIGS. 6A-6F. DP CD8 TIL recognize and kill autologous tumor cells andtheir frequency correlates with improved overall survival. (A) ExpandedCD8 TIL were tested for tumor reactivity by cultivating them for 20 hrswith increasing numbers of autologous tumor cells, and recognition wasassessed by measuring the frequency of 4-1BB expression (filled symbols)and IFN-γ secretion (open symbols). Results are shown for three cancerpatients. (B) Reactivity of DP CD8 TIL was confirmed by culture withautologous tumor cells with or without MHC-I blocking antibody,allogeneic tumor cells, and plate-bound anti-CD3. The up-regulation of4-1BB after 20 hrs is shown for three cancer patients. (C) Tumor cellkilling was measured by assessed the amount of caspase 3/7+ events/wellmonitored every hour over a 20 hrs period. (D) Representative images forDN and DP CD8 TIL taken at the beginning (T=0) and at the end of thecoculture (T=20 hrs). (E) Overall survival on a cohort of 62 HNSCCpatients based on the frequency of DP CD8 TIL among total CD8 TIL at thetime of surgery. (F) Similar analysis performed on a cohort of 30 HPV−negative HNSCC patients.

FIGS. 7A-7C. DP CD8 TIL are enriched in tumor reactive cells and theprimary tumor TCR repertoire overlaps with the metastatic LN TCRrepertoire within the same individual. (A) and (B) DP CD8 TIL weresorted and expanded in vitro. After expansion, DP CD8 TIL werecocultured with autologous tumor cells for 24 hrs. At the end of theculture, cells were sorted based on their expression of 4-1BB and CD25.The TCR repertoire of DP CD8 TIL prior to coculture was compared to theTCR repertoire of 4-1BB+CD25+DP CD8 TIL (tumor-reactive). The top 15clonotypes in the DP CD8 TIL prior to co-culture are present with theirrespective frequencies before and after coculture with tumor cells. Theresults from two cancer patients are indicated. (C) The 500 mostfrequent DP CD8 TIL clonotypes are plotted based on their frequency inthe primary tumor and the metastatic LN. The results from two HNSCCpatients are indicated. SEQ ID NOs: 1-15 are shown in FIG. 7A, in orderfrom top (SEQ ID NO:1) to bottom (SEQ ID NO: 15). SEQ ID NOs: 16-30 areshown in FIG. 7B, in order from top (SEQ ID NO: 16) to bottom (SEQ IDNO: 30).

SEQUENCES

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NOs: 1-30 are the nucleotide sequences of exemplary TCRB CDR3regions.

DETAILED DESCRIPTION

There is a need to improve the efficacy of immunotherapy and adoptive Tcell transfer for cancer patients, and a need to characterize CD8 Tcells involved in the anti-tumor response, in order to use a particularsub-population for diagnostic methods. CD39⁺CD8⁺ T cells includeCD39+CD103+CD8+ T cells and CD39+CD103−CD8+ T cells. It is disclosedherein that the phenotype and function of tumor-reactive CD8 T cells wasdetermined, and it was documented that the co-expression of CD103 andCD39 identified a unique population of tumor-infiltrating CD8 T cellsspecifically induced in the tumor microenvironment. These cells, whichare chronically stimulated within a tumor, are highly enriched for tumorreactivity and have the ability to kill autologous tumor cells. A higherfrequency of CD103+CD39+CD8 T cells in human tumors correlated with agreater overall survival, indicating that the evaluation of these cellsin biological samples provides methods for determining the efficacy oftreatment, and for evaluating whether a subject with a tumor willrespond to treatment.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

4-1BB: A transmembrane protein, also referred to as CD137 and TNFRSF9,that is a protein of the Tumor necrosis factor receptor superfamily(TNFRS). Expression of 4-1BB is generally activation dependent and ispresent in a broad subset of immune cells including activated NK and NKTcells, regulatory T cells, activated CD4 and CD8 T cells, dendriticcells (DC), stimulated mast cells, differentiating myeloid cells,monocytes, neutrophils, and eosinophils (Wang, 2009, ImmunologicalReviews 229: 192-215). 4-1BB expression has also been demonstrated ontumor vasculature (Broll, 2001, Amer. J. Clin. Pathol. I15(4):543-549;Seaman, 2007, Cancer Cell 11: 539-554) and at sites of inflamed oratherosclerotic endothelium (Drenkard, 2007 FASEB J. 21: 456-463;Olofsson, 2008, Circulation 117: 1292-1301). The ligand that stimulates4-1BB, i.e., 4-1BB Ligand (4-1BBL), is expressed on activatedantigen-presenting cells (APCs), myeloid progenitor cells, andhematopoietic stem cells. Human 4-1BB is a 255 amino acid protein (SeeGENBANK Accession Nos. NM-001561 and NP-001552, incorporated herein byreference as available on Jun. 1, 2017). Agonist antibodies of 4-1BB aredisclosed, for example, in U.S. Pat. No. 8,337,850, incorporated hereinby reference.

Altering level: Changing, either by increasing or decreasing, the numberof cells of a specific cell type, or the level of production orexpression of a nucleic acid sequence or an amino acid sequence (forexample a polypeptide, an siRNA, a miRNA, an mRNA, a gene), as comparedto a control level.

Antisense, Sense, and Antigene: DNA has two antiparallel strands, a5′→3′ strand, referred to as the plus strand, and a 3′→5′ strand,referred to as the minus strand. Because RNA polymerase adds nucleicacids in a 5′→3′ direction, the minus strand of the DNA serves as thetemplate for the RNA during transcription. Thus, an RNA transcript willhave a sequence complementary to the minus strand, and identical to theplus strand (except that U is substituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a DNAtarget. An antisense RNA (asRNA) is a molecule of RNA complementary to asense (encoding) nucleic acid molecule.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which recognizes and binds(such as specifically recognizes and specifically binds) an epitope ofan antigen, such as a PD-1, PD-L1, CTLA-4, BTLA, TIM-3, LAG3 or 4-1BBpolypeptide, or a fragment thereof. Immunoglobulin molecules arecomposed of a heavy and a light chain, each of which has a variableregion, termed the variable heavy (V_(H)) region and the variable light(V_(L)) region. Together, the V_(H) region and the V_(L) region areresponsible for binding the antigen recognized by the antibody.

Antibodies include intact immunoglobulins and the variants and portionsof antibodies well known in the art, such as single-domain antibodies(e.g. V_(H) domain antibodies), Fab fragments, Fab′ fragments, F(ab)′₂fragments, single chain Fv proteins (“scFv”), and disulfide stabilizedFv proteins (“dsFv”). A scFv protein is a fusion protein in which alight chain variable region of an immunoglobulin and a heavy chainvariable region of an immunoglobulin are bound by a linker, while indsFvs, the chains have been mutated to introduce a disulfide bond tostabilize the association of the chains. The term also includesgenetically engineered forms such as chimeric antibodies (for example,humanized murine antibodies), heteroconjugate antibodies (such as,bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed.,W. H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (κ). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs has been defined according to Kabat et al.(see, Kabat et al., Sequences of Proteins of Immunological Interest,U.S. Department of Health and Human Services, 1991) and ImMunoGeneTicsdatabase (IMGT) (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; andimgt.cines.fr/IMGT_vquest/vquest?_livret=0&Option=humanIg). The Kabatdatabase is maintained online (ncbi.nlm.nih.gov/igblast/). The sequencesof the framework regions of different light or heavy chains arerelatively conserved within a species, such as humans. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 (or H-CDR3) is located in the variabledomain of the heavy chain of the antibody in which it is found, whereasa V_(L) CDR1 (or L-CDR1) is the CDR1 from the variable domain of thelight chain of the antibody in which it is found. An antibody that bindsPD-1, PD-L1, or PD-L2, for example, will have a specific V_(H) regionand the V_(L) region sequence, and thus specific CDR sequences.Antibodies with different specificities (i.e. different combining sitesfor different antigens) have different CDRs. Although it is the CDRsthat vary from antibody to antibody, only a limited number of amino acidpositions within the CDRs are directly involved in antigen binding.These positions within the CDRs are called specificity determiningresidues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and/or heavy chain genesof a single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds a PD-1,PD-L1, CTLA-4, BTLA, TIM-3, LAG3 or 4-1BB polypeptide.

A “human” antibody (also called a “fully human” antibody) is an antibodythat includes human framework regions and all of the CDRs from a humanimmunoglobulin. In one example, the framework and the CDRs are from thesame originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. A “humanized” immunoglobulin is animmunoglobulin including a human framework region and one or more CDRsfrom a non-human (for example a mouse, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor,” and the human immunoglobulin providing the frameworkis termed an “acceptor.” In one embodiment, all the CDRs are from thedonor immunoglobulin in a humanized immunoglobulin. Constant regionsneed not be present, but if they are, they must be substantiallyidentical to human immunoglobulin constant regions, i.e., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences. A “humanized antibody” is an antibody comprising a humanizedlight chain and a humanized heavy chain immunoglobulin. A humanizedantibody binds to the same antigen as the donor antibody that providesthe CDRs. The acceptor framework of a humanized immunoglobulin orantibody may have a limited number of substitutions by amino acids takenfrom the donor framework. Humanized or other monoclonal antibodies canhave additional conservative amino acid substitutions, which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. Humanized immunoglobulins can be constructed by means ofgenetic engineering (see for example, U.S. Pat. No. 5,585,089).

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. In one embodiment, T cells respond to the epitope, when theepitope is presented in conjunction with an MHC molecule. Epitopes canbe formed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5, about 9, or about 8-10 aminoacids in a unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance.

Antigen-presenting cell (APC): A cell that can present antigen bound toMHC class I or class II molecules to T cells. APCs include, but are notlimited to, monocytes, macrophages, dendritic cells, B cells, T cellsand Langerhans cells. A T cell that can present antigen to other T cells(including CD4+ and/or CD8+ T cells) is an antigen presenting T cell(T-APC).

B- and T-lymphocyte attenuator (BTLA): A protein also known as CD272.BTLA expression is induced during activation of T cells, and BTLAremains expressed on Th1 cells. BTLA interacts with a B7 homolog, B7H4,and plays a role in T-cell inhibition via interaction with tumornecrosis family receptors. BTLA is a ligand for tumor necrosis factor(receptor) superfamily, member 14 (TNFRSF14), also known as herpes virusentry mediator (HVEM). BTLA-HVEM complexes negatively regulate T-cellimmune responses. A specific, non-limiting BTLA amino acid sequence, andan mRNA sequence encoding BTLA, is provided in GENBANK® Accession No.NM_001085357, Sep. 1, 2016, incorporated herein by reference. BTLAantagonists include agents that reduce the expression or activity ofBTLA or inhibits the T-cell inhibition function of BTLA, for example, byspecifically binding to BTLA and inhibiting binding of BTLA to tumornecrosis factor receptors. Exemplary compounds include antibodies (suchas an anti-BTLA antibody), RNAi molecules, antisense molecules, anddominant negative proteins.

Binding or stable binding (oligonucleotide): An oligonucleotide binds orstably binds to a target nucleic acid if a sufficient amount of theoligonucleotide forms base pairs or is hybridized to its target nucleicacid, to permit detection of that binding. Binding can be detected byeither physical or functional properties of the target:oligonucleotidecomplex. Binding between a target and an oligonucleotide can be detectedby any procedure known to one skilled in the art, including bothfunctional and physical binding assays. For instance, binding can bedetected functionally by determining whether binding has an observableeffect upon a biosynthetic process such as expression of a gene, DNAreplication, transcription, translation and the like.

Physical methods of detecting the binding of complementary strands ofDNA or RNA are well known in the art, and include such methods as DNaseI or chemical footprinting, gel shift and affinity cleavage assays,Northern blotting, dot blotting and light absorption detectionprocedures. For example, one method that is widely used, because it issimple and reliable, involves observing a change in light absorption ofa solution containing an oligonucleotide (or an analog) and a targetnucleic acid at 220 to 300 nm as the temperature is slowly increased. Ifthe oligonucleotide or analog has bound to its target, there is a suddenincrease in absorption at a characteristic temperature as theoligonucleotide (or analog) and the target disassociate from each other,or melt.

The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of theoligomer is melted from its target. A higher (T_(m)) means a stronger ormore stable complex relative to a complex with a lower (T_(m)).

Binding affinity: Affinity of an antibody for an antigen. In oneembodiment, affinity is calculated by a modification of the Scatchardmethod described by Frankel et al., Mol. Immunol., 16:101-106, 1979. Inanother embodiment, binding affinity is measured by an antigen/antibodydissociation rate. In another embodiment, a high binding affinity ismeasured by a competition radioimmunoassay. In another embodiment,binding affinity is measured by ELISA. An antibody that “specificallybinds” an antigen (such as a PD-1, PD-L1, CTLA-4, BTLA, TIM-3, LAG3 or4-1BB polypeptide) is an antibody that binds the antigen with highaffinity and does not significantly bind other unrelated antigens.

Cancer therapeutic: Any agent of use for treating cancer in a subject.Cancer therapeutics include a checkpoint inhibitor, biological responsemodifier (for example, cytokines and chemokines), a cancer vaccine,chemotherapy and/or radiation.

Chemotherapeutic agents: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth such as psoriasis. In oneembodiment, a chemotherapeutic agent is a radioactive compound. One ofskill in the art can readily identify a chemotherapeutic agent of use(see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter86 in Harrison's Principles of Internal Medicine, 14th edition; Perry etal., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., ©2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.):Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-YearBook, 1995; Fischer, D. S., Knobf, M. F., Durivage, H. J. (eds): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).Combination chemotherapy is the administration of more than one agent totreat cancer. One example is the administration of an antibody thatbinds PD-1, PD-L1, or CTLA-4 polypeptide used in combination with cells,a radioactive compound, or a chemical compound.

CD39 (ENTPD1): An integral membrane protein with two transmembranedomains and a large extracellular region (Maliszewski et al, 1994). Itwas first identified as an activation marker on human lymphocytes and asthe vascular ecto-ADPase (Kaczmarek E et al. (1996) Biol Chem). The term“CD39” denotes the CD39 protein also named as ectonucleosidetriphosphate diphosphohydrolase-1 (ENTPD1). In vivo CD39 is expressed onregulatory T cells (Treg cells), B cells and several innate immunecells. It plays a key role in immune suppression via hydrolysis ofadenosine triphosphate (ATP) and adenosine diphosphate (ADP) intoadenosine monophosphate (AMP), which is then processed into adenosine byCD73, an ecto-5′-nucleotidase. Adenosine is a potent immunoregulatorthat, via binding to A2A receptors on T cells, enhances the accumulationof intracellular cAMP, thereby preventing T cell activation (Deaglio Set al., JEM, 2007). Expression of both, CD39 and CD73 is increased inseveral human solid malignancies (Antonioli Luca et al., Trends Mol Med,2013). Upregulation of both enzymes is favored within hypoxicenvironments, and their sequential concerted action may play a role intumor immunoescape (Eltzschig H K et al., Blood 2009; Ghiringhelli F etal., J Biomed Biotech, 2012). A specific, non-limiting CD39 amino acidsequence, and an mRNA sequence encoding CD39, is provided in GENBANK®Accession No. NM_001776, May 1, 2017, incorporated herein by reference.

CD103: Known as the integrin alpha E (ITGAE), CD103 binds integrin beta7 (β7-ITGB7) to form the heterodimeric integrin molecule αEβ7. The mainligand for αEβ7 is E-cadherin, an adhesion molecule found on epithelialcells. It is important for T cell homing to the intestinal sites andretention of tissue-resident memory (T_(RM)) cells in tissues. In vivo,CD103 is expressed on a subset of dendritic cells in the gut and apopulation of T cells present on peripheral tissues characterized astissue-resident memory cells (T_(RM)) (Schenkel J M et al., Immunity,2014; Mueller S N et al., Nat Rev Immunol, 2015). CD103 is alsoexpressed on a subset of CD8 T cells in high-grade serous ovariancancer, lung cancer, urothelial cell carcinoma of the bladder andendometrial carcinoma (Webb J R et al., Clin Cancer Res, 2014; Webb J Ret al., Cancer Immunol Res, 2015; Komdeur F L et al., Oncotarget, 2016;Djenidi F et al., J Immunol, 2015; Wang B et al., J Urol, 2015; Workel HH et al, EJC, 2015). In those malignancies, CD103+CD8 T cells arepreferentially localized within the tumor, therefore favoring a directinteraction with tumor cells. CD103+CD8 T cells express high levels ofPD-1, an activation/exhaustion surface molecule, which upon interactionwith its ligand PD-L1 results in inhibition of T cell proliferation,survival and effector functions (Webb J R et al., Cancer Immunol Res,2015). A specific, non-limiting CD103 amino acid sequence, and an mRNAsequence encoding CD103, is provided in GENBANK® Accession No.NM_002208, May 20, 2017, incorporated herein by reference.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease the affinityof a protein, such as an antibody. For example, a human antibody thatspecifically binds PD-1, PD-L1, BTLA, TIM-3, LAG3 CTLA-4 or 4-1BB, caninclude at most about 1, at most about 2, at most about 5, and mostabout 10, or at most about 15 conservative substitutions andspecifically bind the PD-1, PD-L1, BTLA, TIM-3, LAG3, CTLA-4 or 4-1BBpolypeptide. The term conservative variation also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acid,provided that antibody specifically binds the PD-1, PD-L1, BTLA, TIM-3,LAG3, CTLA-4 or 4-1BB polypeptide. Non-conservative substitutions arethose that reduce an activity or binding to a PD-1, PD-L1, BTLA, TIM-3,LAG3, CTLA-4, or 4-1BB polypeptide.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Contacting: Placement in direct physical association; includes both insolid and liquid form.

Control level (immune parameter): A baseline level of an immuneparameter. In some embodiments, and control level is the level of acomponent of the immune system, such as a specific type of cells, in theabsence of a therapeutic agent. A control level can be measured in asample from a subject that has not been treated with an agent ofinterest, or a sample from a subject that has been treated with acontrol agent. The control level can also be a standard value, such as avalue determined from an average of a large number of samples over time.The control level can also be measured in a sample from a subjecttreated with the specific dose of a therapeutic agent, wherein that doseis not administered to the subject at the time the subject is currentlyunder evaluation. The control can be from the subject under evaluation,or can be from a different subject.

Cytotoxic T-lymphocyte-Associated Protein 4 (CTLA-4): A protein alsoknown as CD152. CTLA-4 is a member of the immunoglobulin superfamily.CTLA-4 is a protein receptor that functions as an immune checkpoint, andthus downregulates immune responses. CTLA-4 is constitutively expressedin regulatory T cells (Tregs) and is upregulated in conventional T cellsafter activation. CLTA4 binds CD80 or CD86 on the surface ofantigen-presenting cells, and is an inhibitor of T cells. Specificnon-limiting examples of a CTLA-4 protein and an mRNA encoding CTLA-4are disclosed, for example, in GENBANK® Accession No. NM_001037631, Oct.7, 2016, incorporated herein by reference. CTLA-4 antagonists includeagents that reduce the expression or activity of CTLA-4 or inhibits theT-cell inhibition function of CTLA-4, for example, by specificallybinding to CTLA-4 and inhibiting binding of CTLA-4 to CD80 or CD86 onthe surface of antigen-presenting cells. Exemplary compounds includeantibodies (such as an anti-CTLA-4 antibody), RNAi molecules, antisensemolecules, and dominant negative proteins.

Detecting or detection (cell or biomolecule): Refers to quantitativelyor qualitatively determining the presence of a biomolecule or specificcell type, such as a CD8⁺CD39⁺CD103⁺ T cell, under investigation. Forexample, quantitatively or qualitatively determining the presence ofCD8⁺CD39⁺CD103⁺ T cells in a sample from a subject. Generally, detectionof a biological molecule, such as a protein, nucleic acid, or detectinga specific cell type or cell proliferation, requires performing abiological assay and not simple observation. For example, assays thatutilize antibodies or nucleic acid probes (which can both be labeled),or can be used to detect proteins or cells, respectively. Diagnosing ordiagnosis of the efficacy of treatment, such as with a checkpointinhibitor, involves detecting a significant change in a cell orbiomolecule, such as CD8⁺CD39⁺CD103⁺ T cells.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide, orfor a stop signal. The term codon is also used for the corresponding(and complementary) sequences of three nucleotides in the mRNA intowhich the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intendedto include the reverse complement of that DNA molecule. Except wheresingle-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, a tumor. Diagnostic methodsdiffer in their sensitivity and specificity. The “sensitivity” of adiagnostic assay is the percentage of diseased individuals who testpositive (percent of true positives). The “specificity” of a diagnosticassay is one minus the false positive rate, where the false positiverate is defined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis. “Prognostic” is theprobability of development (e.g., severity) of a pathologic condition,such as a tumor or metastasis.

Encode: A polynucleotide is said to encode a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Feeder Cells: A layer of cells such as on the bottom of a culture dish.The feeder cells can release nutrients, growth factors and/or cytokinesinto the culture medium and provide a substrate to which other cells,such as T cells, can interact. The cells can be irradiated. In oneembodiment, feeder cells are irradiated allogeneic peripheral bloodmononuclear cells.

Immune Checkpoint Inhibitor: A type of agent that blocks biologicalpathways in specific types of immune system cells, such as, but nolimited to, T cells, and some cancer cells. These inhibitors inhibit Tcells from killing cancer cells. When a checkpoint inhibitor is blocked,an “inhibition” on the immune system is reduced and T cells becomeactivated against cancer cells. Examples of checkpoint proteins found onT cells or cancer cells include PD-1, PD-L1, CTLA-4, BTLA, TIM-3.

Immune Response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies.

“Unresponsiveness” with regard to immune cells includes refractivity ofimmune cells to stimulation, such as stimulation via an activatingreceptor or a cytokine. Unresponsiveness can occur, for example, becauseof exposure to immunosuppressants or exposure to high doses of antigen.As used herein, the term “anergy” or “tolerance” includes refractivityto activating receptor-mediated stimulation. Such refractivity isgenerally antigen-specific and persists after exposure to the tolerizingantigen has ceased. For example, anergy in T cells (as opposed tounresponsiveness) is characterized by lack of cytokine production (suchas IL-2). T cell anergy occurs when T cells are exposed to antigen andreceive a first signal (a T cell receptor or CD3 mediated signal) in theabsence of a second signal (a costimulatory signal). Under theseconditions, re-exposure of the cells to the same antigen (even ifexposure occurs in the presence of a costimulatory molecule) results infailure to produce cytokines and, thus, failure to proliferate. AnergicT cells can, however, mount responses to unrelated antigens and canproliferate if cultured with cytokines (such as IL-2). For example, Tcell anergy can also be observed by the lack of IL-2 production by Tlymphocytes as measured by ELISA or by a proliferation assay using anindicator cell line. Alternatively, a reporter gene construct can beused. For example, anergic T cells fail to initiate IL-2 genetranscription induced by a heterologous promoter under the control ofthe 5′ IL-2 gene enhancer or by a multimer of the AP1 sequence that canbe found within the enhancer (Kang et al. Science 257:1134, 1992).Anergic antigen specific T cells may have a reduction of at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% in cytotoxicactivity relative a corresponding control antigen specific T cell.

Inhibiting or treating a disease: Inhibiting a disease, such as tumorgrowth, refers to inhibiting the full development of a disease orlessening the physiological effects of the disease process. In severalexamples, inhibiting or treating a disease refers to lessening symptomsof a tumor or an infection with a pathogen. For example, cancertreatment can prevent the development of paraneoplastic syndrome in aperson who is known to have a cancer, or lessening a sign or symptom ofthe tumor. In another embodiment, treatment of an infection can refer toinhibiting development or lessening a symptom of the infection.“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition related to thedisease. Therapeutic vaccination refers to administration of an agent toa subject already infected with a pathogen. The subject can beasymptomatic, so that the treatment prevents the development of asymptom.

Isolated: An “isolated” biological component, such as a nucleic acid,protein (including antibodies) or cell, has been substantially separatedor purified away from other biological components in the environment(such as other cells) in which the component naturally occurs, i.e.,other chromosomal and extra-chromosomal DNA and RNA, proteins andorganelles. Nucleic acids and proteins that have been “isolated” includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Lymphocyte: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Lymphocyte-activation gene 3 (LAG3): A protein which in humans isencoded by the LAG3 gene, also called CD223. LAG-3 is a cell surfacemolecule with diverse biologic effects on T cell function, and is animmune checkpoint receptor. LAG3 negatively regulates cellularproliferation, activation, and homeostasis of T cells, and has beenreported to play a role in Treg suppressive function. An exemplary aminoacid and mRNA encoding human LAG3 is provided in GENBANK® Accession No.NM_002286.5, Apr. 9, 2017, incorporated herein by reference.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Mean Fluorescence Intensity (flow cytometry): Flow cytometry isconcerned with the measurement of the light intensity of a cell orparticle, whether it be scattered laser light or fluorescence emitted bya fluorochrome. Light is detected by a photomultiplier tube (PMT), whichconverts it via an amplifier to a voltage that is proportional to theoriginal fluorescence intensity and the voltage on the PMT. Thesevoltages, which are a continuous distribution, are converted to adiscrete distribution by an Analog to Digital converter (ADC), whichplaces each signal into a specific channel depending on the level offluorescence. The greater the resolution of the ADC, the closer thisreflects the continuous distribution.

Flow cytometric data can be displayed using either a linear or alogarithmic scale. The use of a logarithmic scale is indicated in mostbiological situations where distributions are skewed to the right. Inthis case the effect is to normalize the distribution—it is said to beLog Normal and the data has been log-transformed. Linear signals comethrough a linear amplifier but the logarithmic transformation may beachieved either by a logarithmic amplifier or by the use of Look UpTables (LUT). Most ADCs in analytical cytometers are 10-bit, i.e., theydivide data into 2e10 or 1024 channels, although there is a growingtrend to use 12- or 14-bit ADCs to give greater resolution of data.

Data from a single data channel (scatter or fluorescence) is displayedas a histogram in which the x axis is divided into 1024 channels (for a10-bit ADC). If the data is in a linear scale, the channel number andthe linear value for that channel will be easily obtained. On alogarithmic scale, the x axis is still divided into 1024 channels but isdisplayed as a 5-log decade scale (in general 5 log decades are used).

To quantify flow cytometric data the measures of the distribution of apopulation are utilized. Generally, the measures of central tendency arethe mean and the median. The mean is the ‘average’ and can be eitherarithmetic or geometric. The arithmetic mean is calculated asSigma(x)/n, and the geometric mean as n root(a1×a2×a3 . . . an). Ingeneral, with log-amplified data the geometric mean is used as it takesinto account the weighting of the data distribution, and the arithmeticmean is used for linear data or data displayed on a linear scale. Themedian is the central value, i.e., the 50th percentile, where half thevalues are above and half below. A cell with “high” expression and “low”expression can be determined relatively depending on the fluorescence ofthe entire population; these parameters are readily visualized on plotsof flow cytometric data.

Medium (tissue culture or cell culture): A synthetic set of cultureconditions with the nutrients necessary to support the growth (cellproliferation/expansion) of a specific population of cells. Mediagenerally include a carbon source, a nitrogen source and a buffer tomaintain pH. In one embodiment, growth medium contains a minimalessential media, such as RPMI, supplemented with various nutrients toenhance cell growth. Additionally, the minimal essential media may besupplemented with additives such as human, calf or fetal bovine serum.

Neoplasia, malignancy, cancer or tumor: A neoplasm is an abnormal growthof tissue or cells that results from excessive cell division. Neoplasticgrowth can produce a tumor. A “cancer” or “tumor” is a neoplasm that hasundergone characteristic anaplasia with loss of differentiation,increase rate of growth, invasion of surrounding tissue, and is capableof metastasis. The amount of a tumor in an individual is the “tumorburden” which can be measured as the number, volume, or weight of thetumor. A tumor that does not metastasize is referred to as “benign.” Atumor that invades the surrounding tissue and/or can metastasize isreferred to as “malignant.” Metastatic cancer is a cancer at one or moresites in the body other than the site of origin of the original(primary) cancer from which the metastatic cancer is derived.

Examples of hematological tumors include leukemias, including acuteleukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors(such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

In several examples, a tumor is a head and neck squamous cell carcinoma,lung cancer, melanoma, ovarian cancer renal cell carcinoma, bladdercancer, cervical cancer, liver cancer, prostate cancer, breast cancer,glioblastoma or rectal cancer.

Parenteral: Administered outside of the intestine, e.g., not via thealimentary tract. Generally, parenteral formulations are those that willbe administered through any possible mode except ingestion. This termespecially refers to injections, whether administered intravenously,intrathecally, intramuscularly, intraperitoneally, intraarticularly, orsubcutaneously, and various surface applications including intranasal,intradermal, and topical application, for instance.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the antibodies herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). A polypeptide can be between 3 and 30 amino acids inlength. In one embodiment, a polypeptide is from about 7 to about 25amino acids in length. In yet another embodiment, a polypeptide is fromabout 8 to about 10 amino acids in length. In yet another embodiment, apeptide is about 9 amino acids in length. With regard to polypeptides,“comprises” indicates that additional amino acid sequence or othermolecules can be included in the molecule, “consists essentially of”indicates that additional amino acid sequences are not included in themolecule, but that other agents (such as labels or chemical compounds)can be included, and “consists of” indicates that additional amino acidsequences and additional agents are not included in the molecule.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease, such as a tumor.“Treating” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop, such as a reduction in tumor burden or a decrease in the numberof size of metastases. “Ameliorating” refers to the reduction in thenumber or severity of signs or symptoms of a disease, such as cancer.

Programmed cell Death protein (PD)-1: PD-1 molecules are members of theimmunoglobulin gene superfamily. The human PD-1 has an extracellularregion containing an immunoglobulin superfamily domain, a transmembranedomain, and an intracellular region including an immunoreceptortyrosine-based inhibitory motif (ITIM) ((Ishida et al., EMBO J. 11:3887,1992; Shinohara et al., Genomics 23:704, 1994; U.S. Pat. No. 5,698,520,incorporated herein by reference). These features also define a largerfamily of molecules, called the immunoinhibitory receptors, which alsoincludes gp49B, PIR-B, and the killer inhibitory receptors (KIRs)(Vivier and Daeron (1997) Immunol. Today 18:286). Without being bound bytheory, it is believed that the tyrosyl phosphorylated ITIM motif ofthese receptors interacts with the S112-domain containing phosphatase,which leads to inhibitory signals. A subset of these immuno-inhibitoryreceptors bind to major histocompatibility complex (MHC) molecules, suchas the KIRs, and cytotoxic T-lymphocyte associated protein 4 (CTLA-4)binds to B7-1 and B7-2. In humans, PD-1 is a 50-55 kDa type Itransmembrane receptor that was originally identified in a T cell lineundergoing activation-induced apoptosis. PD-1 is expressed on T cells, Bcells, and macrophages. The ligands for PD-1 are the B7 family membersPD-ligand 1 (PD-L1, also known as B7-H1) and PD-L2 (also known asB7-DC).

In vivo, PD-1 is expressed on activated T cells, B cells, and monocytes.Experimental data implicates the interactions of PD-1 with its ligandsin down regulation of central and peripheral immune responses. Inparticular, proliferation in wild-type T cells but not in PD-1-deficientT cells is inhibited in the presence of PD-L1. Additionally,PD-1-deficient mice exhibit an autoimmune phenotype. An exemplary aminoacid sequence of human PD-1 is set forth in Ishida et al., EMBO J.11:3887, 1992; Shinohara et al. Genomics 23:704, 1994; U.S. Pat. No.5,698,520):

Engagement of PD-1 (for example by crosslinking or by aggregation),leads to the transmission of an inhibitory signal in an immune cell,resulting in a reduction of immune responses concomitant with anincrease in immune cell anergy. PD-1 binds two ligands, PD-L1 and PD-L2,both of which are human PD-1 ligand polypeptides, that are members ofthe B7 family of polypeptides.

PD-1 antagonists include agents that reduce the expression or activityof a PD ligand 1 (PD-L1) or a PD ligand 2 (PD-L2) or reduces theinteractions between PD-1 and PD-L1, or PD-L2. Exemplary compoundsinclude antibodies (such as an anti-PD-1 antibody, an anti-PD-L1antibody, and an anti-PD-L2 antibody), RNAi molecules (such as anti-PD-1RNAi molecules, anti-PD-L1 RNAi, and an anti-PD-L2 RNAi), antisensemolecules (such as an anti-PD-1 antisense RNA, an anti-PD-L1 antisenseRNA, and an anti-PD-L2 antisense RNA), dominant negative proteins (suchas a dominant negative PD-1 protein, a dominant negative PD-L1 protein,and a dominant negative PD-L2 protein), see, for example, PCTPublication No. 2008/083174, incorporated herein by reference.

Proliferation: The division of a cell to produce progeny, which can bemeasured in a number of ways known in the art. This includes, but is notlimited to, assays that count the total number of cells, assays thatcount the number of cells of a specific cell type, Ki-67 assays,thymidine incorporation, and bromodeoxyuridine assays.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell. Inone embodiment, a preparation is purified such that the protein orpeptide represents at least 50% of the total peptide or protein contentof the preparation. Substantial purification denotes purification fromother proteins or cellular components. A substantially purified proteinis at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific,non-limiting example, a substantially purified protein is 90% free ofother proteins or cellular components.

Sample (Biological sample): Includes biological samples containingfluids, tissues, cells, and subcomponents thereof, such as DNA, RNA, andproteins. For example, common samples include tumor biopsy, bone marrow,spleen, lymph node, blood, e.g., peripheral blood (but can also includeany other source from which CD8⁺CD39⁺CD103⁺ T cells can be isolated,including: tissue biopsy, surgical specimens, fine needle aspirates,autopsy material, and the like).

Specific binding agent: An agent that binds substantially only to adefined target. In one embodiment, the specific binding agent is amonoclonal or polyclonal antibody that specifically binds a PD-1, PD-L1,CTLA-4, BTLA, TIM-3, LAG3 or 4-1BB polypeptide

The term “specifically binds” refers, with respect to an antigen such asPD-1, PD-L1, CTLA-4, BTLA, TIM-3, LAG3 or 4-1BB polypeptide to thepreferential association of an antibody or other ligand, in whole orpart, with a cell or tissue bearing that antigen and not to cells ortissues lacking that antigen. It is, of course, recognized that acertain degree of non-specific interaction may occur between a moleculeand a non-target cell or tissue. Nevertheless, specific binding may bedistinguished as mediated through specific recognition of the antigen.Although selectively reactive antibodies bind antigen, they may do sowith low affinity. Specific binding results in a much strongerassociation between the antibody (or other ligand) and cells bearing theantigen than between the antibody (or other ligand) and cells lackingthe antigen. Specific binding typically results in greater than 2-fold,such as greater than 5-fold, greater than 10-fold, or greater than100-fold increase in amount of bound antibody or other ligand (per unittime) to a cell or tissue bearing the polypeptide as compared to a cellor tissue lacking the polypeptide. Specific binding to a protein undersuch conditions requires an antibody that is selected for itsspecificity for a particular protein. A variety of immunoassay formatsare appropriate for selecting antibodies or other ligands specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow & Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork (1988), for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8⁺ Tcell is a cytotoxic T lymphocyte. In another embodiment, a CD8+ cell isa suppressor T cell. A T cell is “activated” when it can respond to aspecific antigen of interest presented on an antigen presenting cells.

T-cell immunoglobulin and mucin-domain containing-3 (TIM-3): A proteinthat in humans is encoded by the HAVCR2 gene. TIM3 is an immunecheckpoint that is a Th1-specific cell surface protein that regulatesmacrophage activation and enhances the severity of experimentalautoimmune encephalomyelitis in mice. The Tim-3 pathway can interactwith the PD-1 pathway in the exhausted CD8+ T cells in cancer. Anexemplary mRNA and protein sequence for human TIM-3 is provided inGENBANK® Accession No. NM_032782.4, Apr. 30, 2017, incorporated hereinby reference.

Therapeutically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit or suppress growthof a tumor. In one embodiment, a therapeutically effective amount is theamount necessary to eliminate, reduce the size, or prevent metastasis ofa tumor. When administered to a subject, a dosage will generally be usedthat will achieve target tissue concentrations (for example, in tumors)that has been shown to achieve a desired in vitro effect.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A, and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. All GENBANK® Accession numbers are hereinincorporated by reference as they appear in the database on Jun. 1,2017. In case of conflict, the present specification, includingexplanations of terms, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

Methods of Treatment: Adoptive Immunotherapy

Methods are disclosed herein for the treatment of a subject of interest,such as a subject with a tumor. The methods include the administrationof a therapeutically effective amount of CD8⁺CD39⁺CD103⁺ T cells.Methods are disclosed herein for increasing the immune response, such asenhancing the immune system in a subject. Administration of the purifiedCD8⁺CD39⁺CD103⁺ T cells, as disclosed herein, will increase the abilityof a subject to elicit an immune response, such as to a tumor.Therefore, by purifying and generating a purified population of selectedCD8⁺CD39⁺CD103⁺ T cells from a subject ex vivo and introducing atherapeutic amount of these cells, the immune response of the recipientsubject is enhanced. Additional agents can also be administered to thesubject, as discussed below. The subject can be a human or a veterinarysubject.

In one example, the method includes isolating from the donor apopulation of donor cells including CD8⁺CD39⁺CD103⁺ T cells (such asperipheral blood mononuclear cells or T cells from a tumor biopsy), andoptionally expanding the cells. A therapeutically effective amount ofCD8⁺CD39⁺CD103⁺ T cells is administered to the recipient, therebyproducing an immune response to the tumor in the recipient. SuchCD8⁺CD39⁺CD103⁺ T cells can kill cells containing the tumor-associatedantigen or assist other immune cells. In some embodiments, additionalcancer therapeutics, such as chemotherapeutic agents, can beadministered.

In several embodiments the method also includes administering atherapeutically effective amount of a PD-1 antagonist, PD-L1 antagonist,CTLA-4 antagonist, BTLA antagonist, TIM-3 antagonist, LAG3 antagonistand/or a 4-1BB agonist to the subject. The administration of a PD-1antagonist, PD-L1 antagonist, CTLA-4 antagonist, BTLA antagonist, TIM-3antagonist, LAG3 antagonist and/or a 4-1BB agonist is described indetail below. Administration of a therapeutic amount of CD8⁺CD39⁺CD103⁺T cells and a therapeutically effective amount of a PD-1 antagonist,PD-L1 antagonist, CTLA-4 antagonist, BTLA antagonist, TIM-3 antagonist,LAG3 antagonist and/or a 4-1BB agonist can be used prophylactically toprevent recurrence of the tumor in the recipient, or to treat a relapseof the tumor.

Generally, the CD8⁺CD39⁺CD103⁺ T cells are autologous. However, theCD8⁺CD39⁺CD103⁺ T cells can also react to matched MHCs from allogeneicdonors. Generally, the T cells are positive for expression of CD3, CD8,CD39, and CD103. For example, fluorescence activated cell sorting (FACS)can be used to identify (and sort if desired) populations of cells thatare positive for CD3, CD8, CD39 and CD103 by using differently coloredanti-CD3, anti-CD8, anti-CD39 and anti-CD103 antibodies. Briefly, apopulation of cells, such as peripheral blood mononuclear cells or Tcells from a tumor biopsy are incubated in the presence of anti-CD103,anti-CD8, anti-CD39 and optionally anti-CD3 antibodies (each having adifferent fluorophore attached), for a time sufficient for the antibodyto bind to the cells. After removing unbound antibody, cells areanalyzed by FACS using routine methods. In specific examples, theresulting population of CD8⁺CD39⁺CD103⁺ T cells is at least 30% purerelative to the total population of CD8+ positive cells, such as atleast about 50% pure, at least about 60% pure, at least about 70% pure,at least about 80% pure, at least about 90% pure, at least about 95%pure, or at least about 96%, about 97%, about 98% or about 99% purerelative to the total population of CD8 positive cells. Thus, only alimited number of heterologous cells is administered. The cells can beprocessed for more than one round of cell sorting. Populations of Tcells can be tested for mycoplasma, sterility, endotoxin and qualitycontrolled for function and purity prior cryopreservation or prior toinfusion into the recipient.

In one embodiment, labeled antibodies specifically directed to one ormore cell surface markers are used to identify, quantify, and/or isolateCD8⁺CD39⁺CD103⁺ T cells and populations of these cells that expressadditional markers. The antibodies can be conjugated to other compoundsincluding, but not limited to, enzymes, magnetic beads, colloidalmagnetic beads, haptens, fluorochromes, metal compounds, radioactivecompounds or drugs. The enzymes that can be conjugated to the antibodiesinclude, but are not limited to, alkaline phosphatase, peroxidase,urease and ß-galactosidase. The fluorochromes that can be conjugated tothe antibodies include, but are not limited to, fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin,allophycocyanins and Texas Red. For additional fluorochromes that can beconjugated to antibodies see Haugland, R. P., Handbook of FluorescentProbes and Research Products, published by Molecular Probes, 9^(th)Edition (2002). The metal compounds that can be conjugated to theantibodies include, but are not limited to, ferritin, colloidal gold,and particularly, colloidal superparamagnetic beads. The haptens thatcan be conjugated to the antibodies include, but are not limited to,biotin, digoxigenin, oxazalone, and nitrophenol. The radioactivecompounds that can be conjugated or incorporated into the antibodies areknown to the art, and include, but are not limited to, technetium 99(⁹⁹Tc), ¹²⁵I, and amino acids comprising any radionuclides, including,but not limited to, ¹⁴C, ³H and ³⁵S.

In some examples, CD8⁺CD39⁺CD103⁺ T cells are isolated by contacting thecells from a biological sample, such as a peripheral blood sample or atumor sample, with an appropriately labeled antibody. However, othertechniques of differing efficacy may be employed to purify and isolatedesired populations of cells. The separation techniques employed shouldmaximize the retention of viability of the fraction of the cells to becollected. The particular technique employed will, of course, dependupon the efficiency of separation, cytotoxicity of the method, the easeand speed of separation, and what equipment and/or technical skill isrequired.

The data generated by flow-cytometers can be plotted in a singledimension, to produce a histogram, or in two-dimensional dot plots oreven in three dimensions. The regions on these plots can be sequentiallyseparated, based on fluorescence intensity, by creating a series ofsubset extractions, termed “gates.” Specific gating protocols are knownin the art. The plots are generally made on logarithmic scales. Becausedifferent fluorescent dyes' emission spectra overlap, signals at thedetectors are compensated electronically and computationally. Dataaccumulated using the flow cytometer can be analyzed using software suchas FLOWJO® or BD FACSDiva®. The analysis is most often done on aseparate computer. The principles of gating, which allow theidentification of cells that express high or low levels of a protein ofinterest, are well known in the art. Tutorials for learning to establishgates are provided, for example, and the FLOWJO® website. Generally, oneof skill in the art can readily use any FACS machine and computerprograms for data analysis to establish gates to separate cells thatexpress a particular marker. As an example, one of skill in the art canreadily identify cells wherein expression of CD8 is absent (CD8⁻),expression of CD8 is present (CD8⁺).

Additional separation procedures may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents, either joined to a monoclonal antibody or used in conjunctionwith complement, and “panning,” which utilizes a monoclonal antibodyattached to a solid matrix, or another convenient technique. Antibodiesattached to magnetic beads and other solid matrices, such as agarosebeads, polystyrene beads, hollow fiber membranes and plastic Petridishes, allow for direct separation. Cells that are bound by theantibody can be removed from the cell suspension by simply physicallyseparating the solid support from the cell suspension. The exactconditions and duration of incubation of the cells with the solidphase-linked antibodies will depend upon several factors specific to thesystem. The selection of appropriate conditions, however, is well knownin the art.

Unbound cells then can be eluted or washed away with physiologic bufferafter sufficient time has been allowed for the cells expressing a markerof interest (such as, but not limited to, CD8, CD39, CD103, andoptionally CD3, to bind to the solid-phase linked antibodies. The boundcells are then separated from the solid phase by any appropriate method,depending mainly upon the nature of the solid phase and the antibodyemployed, and quantified using methods well known in the art. In onespecific, non-limiting example, bound cells separated from the solidphase are quantified by FACS (see above).

Antibodies may be conjugated to biotin, which then can be removed withavidin or streptavidin bound to a support, or fluorochromes, which canbe used with FACS to enable cell separation and quantitation, as knownin the art.

In another embodiment, an apheresis procedure employing an automatedapheresis instrument (such as the CS-3000 blood cell separator, BaxterHealth Care, Deerfield, Ill., or equivalent machine) can be used tocollect cells from a subject. In a specific, non-limiting example,labeled antibodies specifically directed to one or more cell surfacemarkers are used to identify and quantify the CD8⁺CD39⁺CD103⁺ T cellssuch as the cells disclosed herein.

In some embodiments, the CD8⁺CD39⁺CD103⁺ T cells are expanded in vitroprior to administration to the subject. Expansion methods are disclosedbelow.

The present disclosure also provides therapeutic compositions thatinclude the enriched (such as purified) CD8⁺CD39⁺CD103⁺ T cells andoptionally a PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, BTLAantagonist, TIM-3 antagonist, LAG3 antagonist and/or a 4-1BB agonist.The compositions are of use in the methods disclosed herein, such as,but not limited to, treatment of a subject with a tumor. In particularexamples, a population of CD8⁺CD39⁺CD103⁺ T cells are placed in atherapeutic dose form for administration to a subject in need of them.The PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, BTLAantagonist, TIM-3 antagonist, LAG3 antagonist and/or a 4-1BB agonist isalso present in a therapeutic dose form for administration to a subjectin need of treatment.

A therapeutically effective amount of CD8⁺CD39⁺CD103⁺ T cells isadministered to the subject. Specific, non-limiting examples of atherapeutically effective amount of CD8⁺CD39⁺CD103⁺ T cells includepurified CD8⁺CD39⁺CD103⁺ T cells administered at a dose of about 1×10⁵cells per kilogram of subject to about 1×10⁹ cells per kilogram ofsubject, such as from about 1×10⁶ cells per kilogram to about 1×10⁸cells per kilogram, such as from about 5×10⁶ cells per kilogram to about75×10⁶ cells per kilogram, such as at about 25×10⁶ cells per kilogram,or at about 50×10⁶ cells per kilogram.

Purified CD8⁺CD39⁺CD103⁺ T cells can be administered in single ormultiple doses as determined by a clinician. For example, the cells canbe administered at intervals of approximately one day, two days, threedays, four days, five days, six days, one week, two weeks or monthlydepending on the response desired and the response obtained. In someexamples, once the desired response is obtained, no furtherCD8⁺CD39⁺CD103⁺ T cells are administered. However, if the recipientdisplays one or more symptoms associated with the tumor, atherapeutically effective amount of CD8⁺CD39⁺CD103⁺ T cells can beadministered at that time.

The administration can be local or systemic. In some embodiments, thecells are administered intravenously after the subject is treated inchemotherapy. In other embodiments the subject is also administeredcytokines, such as IL-2 and/or IL-15, to support proliferation of theadministered cells.

The purified CD8⁺CD39⁺CD103⁺ T cells disclosed herein can beadministered with a pharmaceutically acceptable carrier, such as saline.The PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, BTLAantagonist, TIM-3 antagonist, LAG3 antagonist and/or a 4-1BB agonist canalso be formulated in a pharmaceutically acceptable carrier, asdescribed below. These can be formulated in a single composition, or intwo separate compositions. In some examples, other therapeutic agentsare administered with the T cells. Other therapeutic agents can beadministered before, during, or after administration of theCD8⁺CD39⁺CD103⁺ T cells, depending on the desired effect. Exemplarytherapeutic agents include, but are not limited to, anti-microbialagents, immune stimulants such as interferon-alpha, chemotherapeuticagents or peptide vaccines used to stimulate T cells in vitro. In aparticular example, compositions containing CD8⁺CD39⁺CD103⁺ T cells alsoinclude the one or more therapeutic agents. The use of CD8⁺CD39⁺CD103⁺ Tcells can reduce tumor volume, tumor metastasis, tumor reoccurrence.

Generally, the methods include selecting a subject having a tumor, suchas a benign or malignant tumor, and administering to the subject atherapeutically effective amount of (1) CD8⁺CD39⁺CD103⁺ T cells and (2)optionally a checkpoint inhibitor antagonist, such as a PD-1 antagonist,a PD-L1 antagonist, a BTLA antagonist, a TIM-3 antagonist, a LAG3antagonist, or a CTLA-4 antagonist, or a 4-1BB agonist. The PD-1antagonist, PD-L1 antagonist, BTLA antagonist, TIM-3 antagonist, LAG3antagonist or CTLA-4 antagonist, or the 4-1BB agonist, can, in somenon-limiting examples, be an antibody (or antigen binding fragmentthereof) that specifically binds PD-1, PD-L1, PD-L1, PD-L2, TIM-3, LAG3,BTLA, CTLA-4, or 4-1BB, respectively. The CD8⁺CD39⁺CD103⁺ T cells are ofuse for treating the tumor, such as for reducing tumor volume, reducingor preventing metastasis, preventing the conversion of a benign to amalignant tumor and/or preventing or inhibiting reoccurrence of thetumor. The administration can be local or systemic. Suitableadministration methods are known to a clinician.

In some embodiments, an advantage of the methods provided herein is thatthe combination of CD8⁺CD39⁺CD103⁺ T cells with checkpoint inhibitorssuch as a PD-1 antagonist, PD-L1 antagonist, BTLA antagonist, TIM-3antagonist, LAG3 antagonist and/or a CTLA-4 antagonist, or a 4-1BBagonist, allows for reduced dosage of active agents for cancer therapy,while also reducing any corresponding undesired side-effects (such ascytotoxicity) of the therapy. In further embodiments, another advantageof the methods provided herein is that that the combination ofCD8⁺CD39⁺CD103⁺ T cells with checkpoint inhibitors such as a PD-1antagonist, PD-L1 antagonist, BTLA antagonist, TIM-3 antagonist, LAG3antagonist and/or a CTLA-4 antagonist, or with a 4-1BB agonist, fortreating the tumor, such as for reducing tumor volume, reducing orpreventing metastasis, preventing the conversion of a benign to amalignant tumor and/or preventing or inhibiting reoccurrence of thetumor. In additional embodiments, the combination of CD8⁺CD39⁺CD103⁺ Tcells with checkpoint inhibitors such as a PD-1 antagonist, PD-L1antagonist, BTLA antagonist, TIM-3 antagonist, LAG3 antagonist and/or aCTLA-4 antagonist, or a 4-1BB agonist allows for increased survival.

Additional agents can also be administered to the subject of interest,such as, but not limited to, cancer therapeutics. Additional treatmentscan also be administered to the subject, such as, but not limited to,surgical resection of the tumor.

The subject can be selected for treatment. For example, a diagnosticassay (such as an immunohistochemical (IHC) assay can be performed onthe tumor (or a sample on the tumor) to identify the subject as onelikely to respond to the disclosed method of treatment. Methods ofselection are disclosed below.

In further embodiments, the subject is selected for treatment if thetumor tests positive for PD-L1 or PD-L2 expression by an IHC assay.Exemplary assays for detecting a tumor that tests positive for PD-L1expression are provided in Topalian et al. 2012. Safety, activity, andimmune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med.366:2443-2454; Wolchok et al. 2013. Nivolumab plus ipilimumab inadvanced melanoma. N. Engl. J. Med. 369:122-133; Herbst et al. 2014.Predictive correlates of response to the anti-PD-L1 antibody MPDL3280Ain cancer patients. Nature. 515:563-567; Garon et al. 2015.Pembrolizumab for the treatment of non-small-cell lung cancer. N. Engl.J. Med. 372:2018-2028; and Reck et al. Pembrolizumab versus chemotherapyfor PD-L1-positive non-small-cell lung cancer. N. Engl. J. Med.375:1823-1833, each of which is incorporated by reference herein.

The tumor can be benign or malignant. The tumor can be any tumor ofinterest, including, but not limited to, a head and neck squamous cellcarcinoma, lung cancer, melanoma, ovarian cancer renal cell carcinoma,bladder cancer, cervical cancer, liver cancer, prostate cancer, breastcancer, glioblastoma or rectal cancer. The lung cancer can be small cellor non-small cell carcinoma of the lung. The liver cancer can be ahepatic carcinoma. The breast cancer can be triple negative breastcancer. In some embodiments, the tumor is a head and neck tumor, such astumors of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity,oropharynx, larynx, hypopharynx, salivary glands and paragangliomas.Additional examples are skin tumors, brain tumors, cervical carcinomas,testicular carcinomas, gastrointestinal tract tumors, genitourinarysystem tumors, gynecological system tumors, endocrine system tumors, asarcoma of the soft tissue and bone, a mesothelioma, a melanoma, aneoplasm of the central nervous system, or a leukemia. In otherembodiments, the tumor is a lung tumor, such as a non-small cell lungcancer or a small cell lung cancer. In further embodiments, the tumorcan be a tumor of the gastrointestinal tract, such as cancer of theesophagus, stomach, pancreas, liver, biliary tree, small intestine,colon, rectum and anal region. In yet other embodiments, the tumor canbe a tumor of the genitourinary system, such as cancer of the kidney,urethra, bladder, prostate, urethra, penis and testis. In someembodiments, the tumor is a gynecologic tumor, such as cancer of thecervix, vagina, vulva, uterine body, gestational trophoblastic diseases,ovarian, fallopian tube, peritoneal, or breast. In other embodiments,the tumor is an endocrine system tumor, such as a thyroid tumor,parathyroid tumor, adrenal cortex tumor, pancreatic endocrine tumor,carcinoid tumor and carcinoid syndrome. The tumor can be a sarcoma ofthe soft tissue and bone, a mesothelioma, a cancer of the skin, amelanoma, comprising cutaneous melanomas and intraocular melanomas, aneoplasm of the central nervous system, a cancer of the childhood,comprising retinoblastoma, Wilm's tumor, neurofibromatoses,neuroblastoma, Ewing's sarcoma family of tumors, rhabdomyosarcoma. Thetumor can be a lymphoma, comprising non-Hodgkin's lymphomas, cutaneousT-cell lymphomas, primary central nervous system lymphoma, and Hodgkin'sdisease. The tumor can be a leukemia, such as acute leukemia, chronicmyelogenous leukemia and lymphocytic leukemia. The tumor can be plasmacell neoplasms, a cancer of unknown primary site, a peritonealcarcinomastosis, a Kaposi's sarcoma, AIDS-associated lymphomas,AIDS-associated primary central nervous system lymphoma, AIDS-associatedHodgkin's disease and AIDS-associated anogenital cancers, a metastaticcancer to the liver, metastatic cancer to the bone, malignant pleuraland pericardial effusions and malignant ascites.

In some embodiments, treatment of the tumor is initiated after thediagnosis of the tumor, or after the initiation of a precursor condition(such as dysplasia or development of a benign tumor). Treatment can beinitiated at the early stages of cancer, for instance, can be initiatedbefore a subject manifests symptoms of a condition, such as during astage I diagnosis or at the time dysplasia is diagnosed or an in situproliferative condition is diagnosed. However, treatment can beinitiated during any stage of the disease, such as but not limited tostage I, stage II, stage III and stage IV cancers. In some examples,treatment is administered to these subjects with a benign tumor that canconvert into a malignant or even metastatic tumor.

Treatment prior to the development of the condition, such as treatmentupon detecting dysplasia or an early (benign) precursor condition, isreferred to herein as treatment of a subject that is “at risk” ofdeveloping the condition. In some embodiments, administration of acomposition can be performed during or after the occurrence of theconditions described herein. The compositions can be administered to asubject at risk of developing the tumor.

The presence of a tumor can be determined by methods known in the art,and typically include cytological and morphological evaluation. Thetumor can be an established tumor. The cells can be in vivo or ex vivo,including cells obtained from a biopsy.

Treatment initiated after the development of a condition, such asmalignant cancer, may result in decreasing the severity of the symptomsof one of the conditions, or completely removing the symptoms, orreducing metastasis, tumor volume or number of tumors. In some example,the tumor becomes undetectable following treatment.

In one aspect of the disclosure, the formation of tumors, such asmetastasis, is delayed, prevented or decreased. In another aspect, thesize of the primary tumor is decreased. In a further aspect, a symptomof the tumor is decreased. In yet another aspect, tumor volume isdecreased. In yet another aspect reoccurrence of the tumor is delayed orprevented, such as for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 2, 22, 23, or 24 months, or for 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 years.

In some embodiments immune response can be measured, tumor volume can bemeasured, the number of metastatic lesions can be measured, and/or asymptom of a tumor can be measured. A therapeutically effective dose canincrease the immune response, decrease tumor volume, decrease the numberand/or size of metastases, and/or decrease one or more symptoms of thetumor.

While the disclosed methods and compositions will typically be used totreat human subjects they may also be used to treat similar or identicaldiseases in other vertebrates, such as other primates, dogs, cats,horses, and cows. A suitable administration format may best bedetermined by a medical practitioner for each subject individually.Various pharmaceutically acceptable carriers and their formulation aredescribed in standard formulation treatises, e.g., Remington'sPharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. andHanson, M. A., Journal of Parenteral Science and Technology, TechnicalReport No. 10, Supp. 42: 2S, 1988. The dosage form of the pharmaceuticalcomposition will be determined by the mode of administration chosen.

CD8⁺CD39⁺CD103⁺ T cells can be administered locally or systemically, byany route. For example, CD8⁺CD39⁺CD103⁺ T cells can be administeredintratumorally, intraperitoneally, intravenously. In one non-limitingexample, the CD8⁺CD39⁺CD103⁺ T cells can be administered intravenously.A PD-1, PD-L1, PD-L2, BTLA, TIM-3, LAG3, or CTLA-4 antagonist (or a4-1BB agonist) also can be administered by any route, includingparenteral administration, for example, intravenous, intraperitoneal,intramuscular, intraperitoneal, intrasternal, or intraarticularinjection or infusion, or by sublingual, oral, topical, intranasal, ortransmucosal administration, or by pulmonary inhalation. In someembodiments, the CD8⁺CD39⁺CD103⁺ T cells and/or the PD-1, PD-L1, PD-L2,BTLA, TIM-3, LAG3, or CTLA-4 antagonist are administered to a tissuewherein the tumor is located, or directly into the tumor (intratumoral).When a parenteral composition is provided, e.g. for injection orinfusion, active agents are generally suspended in an aqueous carrier,for example, in an isotonic buffer solution at a pH of about 3.0 toabout 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or3.5 to about 5.0. Useful buffers include sodium citrate-citric acid andsodium phosphate-phosphoric acid, and sodium acetate-acetic acidbuffers. A form of repository or “depot” slow release preparation may beused so that therapeutically effective amounts of the preparation aredelivered into the bloodstream over many hours or days followingtransdermal injection or delivery.

In certain embodiments, the PD-1, PD-L1, PD-L2, BTLA, TIM-3, LAG3, orCTLA-4 antagonist (such as, but not limited to, an antibody or antigenbinding fragment), or the 4-1BB agonist can be administered at a dose inthe range of from about 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg,0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg,0.05-0.5 mg/kg, according to a dosing schedule of administrationincluding without limitation daily, 2 or 3 times per week, weekly, every2 weeks, every 3 weeks, monthly, etc., or other dose and dosing scheduledeemed appropriate by the treating physician. As part of the combinationtherapy, the CD8⁺CD39⁺CD103⁺ T cells can be administered to the subjectbefore, after, or concurrent with the additional agent, as long as theadministration schedule provides for a sufficient physiologicalconcentrations of the agents to provide a therapeutic benefit.

In some embodiments, the PD-1 or PD-L1 antagonist (such as an antibodyor antigen binding fragment that specifically binds to PD-1 or PD-L1)can be administered at a dose in the range of from about 0.01-10 mg/kg,0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosing schedule ofadministration including without limitation daily, 2 or 3 times perweek, weekly, every 2 weeks, every 3 weeks, monthly, etc., or other doseand dosing schedule deemed appropriate by the treating physician. Aspart of the combination therapy, the CD8⁺CD39⁺CD103⁺ T cells can beadministered to the subject before, after, or concurrent to the PD-1 orPD-L1 antagonist, as long as the administration schedule provides for asufficient physiological concentrations of the agents to provide atherapeutic benefit. In certain embodiments, the CTLA-4 antagonist (suchas an antibody or antigen binding fragment that specifically binds toCTLA-4) can be administered at a dose in the range of from about 0.01-10mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosingschedule of administration including without limitation daily, 2 or 3times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc., orother dose and dosing schedule deemed appropriate by the treatingphysician. As part of the combination therapy, the CD8⁺CD39⁺CD103⁺ Tcells can be administered to the subject before, after, or concurrent tothe CTLA-4 antagonist, as long as the administration schedule providesfor a sufficient physiological concentrations of the agents to provide atherapeutic benefit. In further embodiments, the BTLA antagonist (suchas an antibody or antigen binding fragment that specifically binds toBTLA) can be administered at a dose in the range of from about 0.01-10mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosingschedule of administration including without limitation daily, 2 or 3times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc., orother dose and dosing schedule deemed appropriate by the treatingphysician. As part of the combination therapy, the CD8⁺CD39⁺CD103⁺ Tcells can be administered to the subject before, after, or concurrent tothe BTLA antagonist, as long as the administration schedule provides fora sufficient physiological concentrations of the agents to provide atherapeutic benefit. In additional embodiments, the LAG3 or TIM-3antagonist (such as an antibody or antigen binding fragment thatspecifically binds to LAG3 or TIM-3) can be administered at a dose inthe range of from about 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg,0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg,0.05-0.5 mg/kg, according to a dosing schedule of administrationincluding without limitation daily, 2 or 3 times per week, weekly, every2 weeks, every 3 weeks, monthly, etc., or other dose and dosing scheduledeemed appropriate by the treating physician. As part of the combinationtherapy, the CD8⁺CD39⁺CD103⁺ T cells can be administered to the subjectbefore, after, or concurrent to the LAG3 or TIM-3 antagonist, as long asthe administration schedule provides for a sufficient physiologicalconcentrations of the agents to provide a therapeutic benefit. In yetother embodiments, the 4-1BB agonist (such as an antibody or antigenbinding fragment that specifically binds to 4-1BB) can be administeredat a dose in the range of from about 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg,0.05-0.5 mg/kg, according to a dosing schedule of administrationincluding without limitation daily, 2 or 3 times per week, weekly, every2 weeks, every 3 weeks, monthly, etc., or other dose and dosing scheduledeemed appropriate by the treating physician. As part of the combinationtherapy, the CD8⁺CD39⁺CD103⁺ T cells can be administered to the subjectbefore, after, or concurrent to the 4-1BB agonist, as long as theadministration schedule provides for a sufficient physiologicalconcentrations of the agents to provide a therapeutic benefit.

Sustained release compositions can be utilized. Suitable examples ofsustained-release compositions include suitable polymeric materials(such as, for example, semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules), suitable hydrophobicmaterials (such as, for example, an emulsion in an acceptable oil) orion exchange resins, and sparingly soluble derivatives (such as, forexample, a sparingly soluble salt). Sustained-release formulations maybe administered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,gels, drops or transdermal patch), bucally, or as an oral or nasalspray, depending on the location of the tumor.

The pharmaceutically acceptable carriers and excipients useful in thedisclosed methods are conventional. For instance, parenteralformulations usually comprise injectable fluids that arepharmaceutically and physiologically acceptable fluid vehicles such aswater, physiological saline, other balanced salt solutions, aqueousdextrose, glycerol or the like. Excipients that can be included are, forinstance, proteins, such as human serum albumin or plasma preparations.If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of non-toxic auxiliary substances, such as wettingor emulsifying agents, preservatives, and pH buffering agents and thelike, for example sodium acetate or sorbitan monolaurate. Actual methodsof preparing such dosage forms are known, or will be apparent, to thoseskilled in the art.

Kits are also provided. CD8⁺CD39⁺CD103⁺ T cells and/or the PD-1, PD-L1,PD-L2, LAG3, TIM-3, BTLA, or CTLA-4 antagonist can be formulated in unitdosage form, suitable for individual administration of precise dosages.The amount of active compound(s) administered will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in amounts effective toachieve the desired effect in the subject being treated. Multipletreatments are envisioned, such as over defined intervals of time, suchas daily, bi-weekly, weekly, bi-monthly or monthly, such that chronicadministration is achieved.

Additional agents can be administered, such as a cytokine, a chemokine,or a chemotherapeutic agent. These can be included in the disclosedpharmaceutical compositions. A cytokine can be administered, such asinterleukin-2 (IL-2), granulocyte macrophage colony stimulating factor(GM-CSF), or interferon, such as interferon (IFN) β. In one example, forthe prevention and treatment of cancer, surgical treatment can beadministered to the subject. In one example, this administration issequential. In other examples, this administration is simultaneous.

Examples of chemotherapeutic agents are alkylating agents,antimetabolites, natural products, or hormones and their antagonists.Examples of alkylating agents include nitrogen mustards (such asmechlorethamine, cyclophosphamide, melphalan, uracil mustard orchlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (suchas carmustine, lomustine, semustine, streptozocin, or dacarbazine).Examples of antimetabolites include folic acid analogs (such asmethotrexate), pyrimidine analogs (such as 5-FU or cytarabine), andpurine analogs, such as mercaptopurine or thioguanine. Examples ofnatural products include vinca alkaloids (such as vinblastine,vincristine, or vindesine), epipodophyllotoxins (such as etoposide orteniposide), antibiotics (such as dactinomycin, daunorubicin,doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (suchas L-asparaginase). Examples of miscellaneous agents include platinumcoordination complexes (such as cis-diamine-dichloroplatinum II alsoknown as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocroticalsuppressants (such as mitotane and aminoglutethimide). Examples ofhormones and antagonists include adrenocorticosteroids (such asprednisone), progestins (such as hydroxyprogesterone caproate,medroxyprogesterone acetate, and magestrol acetate), estrogens (such asdiethylstilbestrol and ethinyl estradiol), antiestrogens (such astamoxifen), and androgens (such as testerone proprionate andfluoxymesterone). Examples of the most commonly used chemotherapy drugsinclude Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU,Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, suchas docetaxel), Velban, Vincristine, VP-16, while some more newer drugsinclude Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11),Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin),Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples ofimmunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.),bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocytemacrophage colony stimulating factor; Genetics Institute), IL-2 (Cetusor Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG(from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosisfactor; Genentech). In some embodiments, the subject is administeredsorafenib.

Additional chemotherapeutic agent can be an antibody. Antibodies may beprovided in lyophilized form and rehydrated with sterile water beforeadministration, although they are also provided in sterile solutions ofknown concentration. The antibody solution is then added to an infusionbag containing 0.9% sodium chloride, USP, and typically administered ata dosage of from 0.5 to 15 mg/kg of body weight. Considerable experienceis available in the art in the administration of antibody drugs, whichhave been marketed in the U.S. since the approval of RITUXAN® in 1997.The antibody can specifically bind programmed death (PD)-1 or programmeddeath ligand (PD-L1) (see below). Antibodies can be administered by slowinfusion, rather than in an intravenous push or bolus. In one example, ahigher loading dose is administered, with subsequent, maintenance dosesbeing administered at a lower level. For example, an initial loadingdose of 4 mg/kg may be infused over a period of some 90 minutes,followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infusedover a 30 minute period if the previous dose was well tolerated.

Treatment regimens also include combination with surgery, chemotherapy,radiation, or other immunoablative agents such as CAMPATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludarabine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. peptide vaccine, such as that described inIzumoto et al. 2008 J Neurosurg 108:963-971. Exemplary chemotherapeuticagents include an anthracycline (e.g., doxorubicin (e.g., liposomaldoxorubicin)). a vinca alkaloid (e.g., vinblastine, vincristine,vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide,decarbazine, melphalan, ifosfamide, temozolomide), an immune cellantibody (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), anantimetabolite (including, e.g., folic acid antagonists, pyrimidineanalogs, purine analogs and adenosine deaminase inhibitors (e.g.,fludarabine)), an mTOR inhibitor, a TNFR glucocorticoid induced TNFRrelated protein (GITR) agonist, a proteasome inhibitor (e.g.,aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such asthalidomide or a thalidomide derivative (e.g., lenalidomide).

General Chemotherapeutic agents considered for use in combinationtherapies include anastrozole (ARIMIDEX®), bicalutamide (CASODEX®),bleomycin sulfate (BLENOXANE®), busulfan (MYLERAN®), busulfan injection(BUSULFEX®), capecitabine (XELODA®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (PARAPLATIN®),carmustine (BICNU®), chlorambucil (LEUKERAN®), cisplatin (PLATINOL®),cladribine (LEUSTATIN®), cyclophosphamide (CYTOXAN® or NEOSAR®),cytarabine, cytosine arabinoside (CYTOSAR-U®), cytarabine liposomeinjection (DEPOCYT®), dacarbazine (DTIC-DOME®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (CERUBIDINE®),daunorubicin citrate liposome injection (DAUNOXOME®), dexamethasone,docetaxel (TAXOTERE®), doxorubicin hydrochloride (ADRIAMYCIN®, RUBEX®),etoposide (VEPESID®), fludarabine phosphate (FLUDARA®), 5-fluorouracil(ADRUCIL®, EFUDEX®), flutamide (EULEXIN®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (HYDREA®), Idarubicin (IDAMYCIN®),ifosfamide (IFEX®), irinotecan (CAMPTOSAR®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (ALKERAN®), 6-mercaptopurine(PURINETHOL®), methotrexate (FOLEX®), mitoxantrone (NOVANTRONE®),mylotarg, paclitaxel (TAXOL®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (GLIADEL®), tamoxifen citrate(NOLVADEX®), teniposide (VUMON®), 6-thioguanine, thiotepa, tirapazamine(TIRAZONE®), topotecan hydrochloride for injection (HYCAMPTIN®),vinblastine (VELBAN®), vincristine (ONCOVIN®), and vinorelbine(NAVELBINE®). Exemplary alkylating agents include, without limitation,nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,nitrosoureas and triazenes): uracil mustard (AMINOURACIL MUSTARD®,CHLORETHAMINACIL®, DEMETHYLDOPAN®, DESMETHYLDOPAN®, HAEMANTHAMINE®,NORDOPAN®, URACIL NITROGEN MUSTARD®, URACILLOST®, URACILMOSTAZA®,URAMUSTIN®, URAMUSTINE®), chlormethine (MUSTARGEN®), cyclophosphamide(CYTOXAN®, NEOSAR®, CLAFEN®, ENDOXAN®, PROCYTOX®, REVIMMUNE™),ifosfamide (MITOXANA®), melphalan (ALKERAN®), Chlorambucil (LEUKERAN®),pipobroman (AMEDEL®, VERCYTE®), triethylenemelamine (HEMEL®, HEXYLEN®,HEXASTAT®), triethylenethiophosphoramine, Temozolomide (TEMODAR®),thiotepa (THIOPLEX®), busulfan (BUSILVEX®, MYLERAN®), carmustine(BiCNU®), lomustine (CEENU®), streptozocin (ZANOSAR®), and Dacarbazine(DTIC-DOME®). Additional exemplary alkylating agents include, withoutlimitation, Oxaliplatin (ELOXATIN®); Temozolomide (TEMODAR® andTEMODAL®); Dactinomycin (also known as actinomycin-D, COSMEGEN®);Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard.ALKERAN®); Altretamine (also known as hexamethylmelamine (HMM),HEXYLEN®); Carmustine (BICNU®); Bendamustine (TREANDA®); Busulfan(BUSULFEX® and MYLERAN®); Carboplatin (PARAPLATIN®); Lomustine (alsoknown as CCNU, CEENU®); Cisplatin (also known as CDDP, PLATINOL® andPLATINOL®-AQ); Chlorambucil (LEUKERAN®); Cyclophosphamide (CYTOXAN® andNEOSAR®); Dacarbazine (also known as DTIC, DIC and imidazolecarboxamide, DTIC-DOME®); Altretamine (also known as hexamethylmelamine(HMM), HEXYLEN®); Ifosfamide (IFEX®); Prednumustine; Procarbazine(MATULANE®); Mechlorethamine (also known as nitrogen mustard, mustineand mechloroethamine hydrochloride, MUSTARGEN®); Streptozocin(ZANOSAR®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA,THIOPLEX®); Cyclophosphamide (ENDOXAN®, CYTOXAN®, NEOSAR®, PROCYTOX®,REVIMMUNE®); and Bendamustine HCl (TREANDA®). Exemplary mTOR inhibitorsinclude, e.g., temsirolimus; ridaforolimus (formally known asdeferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); everolimus (AFINITOR® or RAD001);rapamycin (AY22989, SIROLIMUS®); simapimod (CAS164301-51-3);emsirolimus,(5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-,inner salt (SF1126, CAS 936487-67-1), and XL765. Exemplaryimmunomodulators include, e.g., afutuzumab (available from ROCHE®);pegfilgrastim (NEULASTA®); lenalidomide (CC-5013, REVLIMID®);thalidomide (THALOMID®), actimid (CC4047); and IRX-2 (mixture of humancytokines including interleukin 1, interleukin 2, and interferon γ, CAS951209-71-5, available from IRX Therapeutics). Exemplary anthracyclinesinclude, e.g., doxorubicin (Adriamycin® and RUBEX); bleomycin(LENOXANE®); daunorubicin (dauorubicin hydrochloride, daunomycin, andrubidomycin hydrochloride, CERUBIDINE®); daunorubicin liposomal(daunorubicin citrate liposome, DAUNOXOME®); mitoxantrone (DHAD,NOVANTRONE®); epirubicin (ELLENCE™); idarubicin (IDAMYCIN®, IDAMYCINPFS®); mitomycin C (MUTAMYCIN®); geldanamycin; herbimycin; ravidomycin;and desacetylravidomycin. Exemplary vinca alkaloids include, e.g.,vinorelbine tartrate (NAVELBINE®), Vincristine (ONCOVIN®), and Vindesine(ELDISINE®)); vinblastine (also known as vinblastine sulfate,vincaleukoblastine and VLB, ALKABAN-AQ® and VELBAN®); and vinorelbine(NAVELBINE®). Exemplary proteosome inhibitors include bortezomib(VELCADE®); carfilzomib (PX-171-007,(S)-4-Methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide);marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib(CEP-18770); andO-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide(ONX-0912). Exemplary GITR agonists include, e.g., GITR fusion proteinsand anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as,e.g., a GITR fusion protein described in U.S. Pat. No. 6,111,090,European Patent No.: 090505B1, U.S. Pat. No. 8,586,023, PCT PublicationNos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibodydescribed, e.g., in U.S. Pat. No. 7,025,962, European Patent No.1947183B1, U.S. Pat. Nos. 7,812,135, 8,388,967, 8,591,886, EuropeanPatent No. EP 1866339, PCT Publication No. WO 2011/028683, PCTPublication No. WO 2013/039954, PCT Publication No. WO2005/007190, PCTPublication No. WO 2007/133822, PCT Publication No. WO2005/055808. PCTPublication No. WO 1999/40196. PCT Publication No. WO 2001/03720. PCTPublication No. WO 1999/20758, PCT Publication No. WO2006/083289, PCTPublication No. WO 2005/115451, U.S. Pat. No. 7,618,632, and PCTPublication No. WO 2011/051726.

Method of Expanding CD39+CD103+CD8+ T Cells

CD8⁺CD39⁺CD103⁺ T cells can be expanded in vitro. In some embodiments,CD8⁺CD39⁺CD103⁺ T cells isolated from a subject can be cultured intissue culture medium comprising glutamine, serum, and antibiotics toform primary cultures. The cells are generally seeded in an appropriateculture vessel. A culture vessel used for culturing the cell(s) caninclude, but is particularly not limited to: flask, flask for tissueculture, dish, petri dish, dish for tissue culture, multi dish, microplate, micro-well plate, multi plate, multi-well plate, micro slide,chamber slide, tube, tray, CELLSTACK® Chambers, culture bag, and rollerbottle, as long as it is capable of culturing the T cells therein. Thecells can be cultured in a volume of at least or about 0.2, 0.5, 1, 2,5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, orany range derivable therein, depending on the needs of the culture. Insome embodiments, the culture vessel can be a tissue culture plate, forexample, a 6-well, 24-well, or 96-well plate. In other embodiments, theculture vessel can be a bioreactor, which may refer to any device orsystem ex vivo that supports a biologically active environment such thatcells can be propagated. The bioreactor can have a volume of at least orabout 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters,1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein, canbe cultured with the nutrients necessary to support the growth of thepopulation of cells.

Generally, the cells are cultured in growth media including a carbonsource, a nitrogen source and a buffer to maintain pH. The medium canalso contain fatty acids or lipids, amino acids (such as non-essentialamino acids), vitamin(s), growth factors, cytokines, antioxidantsubstances, pyruvic acid, buffering agents, and inorganic salts. Anexemplary growth medium contains a minimal essential media, such asDulbecco's Modified Eagle's medium (DMEM) or ESSENTIAL 8™ (E8™) medium,supplemented with various nutrients, such as non-essential amino acidsand vitamins, to enhance T cell growth. Examples of minimal essentialmedia include, but are not limited to, Minimal Essential Medium Eagle(MEM) Alpha medium, Dulbecco's modified Eagle medium (DMEM), RoswellPark Memorial Institute (RPMI)-1640 medium, 199 medium, and F12 medium.Optionally, antibiotics can be added to a medium, such as, but notlimited to, penicillin, streptomycin, or tetracycline. Glutamine canalso be added to a tissue culture medium. Additives such as antibioticsand amino acids are known in the art.

Additionally, the minimal essential media may be supplemented withadditional additives such as human, fetal calf or bovine serum. Serumcan be included, for example, at a concentration of 10-15%(volume/volume), such as at about 5%, about 6%, about 7%, about 8%,about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, orabout 15% serum. The serum can be fetal calf serum. In otherembodiments, the serum is human serum, such as human AB serum.

Alternatively, the medium can be serum free. In other cases, the growthmedia may contain a serum replacement. Exemplary serum replacements areknown in the art. For example, KNOCKOUT™ serum replacement is disclosed,for example, in U.S. Patent Application No. 2002/0076747, which isincorporated herein by reference. Alternatives to serum can includematerials which appropriately contain albumin (such as lipid-richalbumin, albumin substitutes such as recombinant albumin, plant starch,dextrans and protein hydrolysates), transferrin (or other irontransporters), fatty acids, insulin, collagen precursors, traceelements, 2-mercaptoethanol, 3′-thiolgiycerol, or equivalents thereto.The alternatives to serum can be prepared by the method disclosed inInternational Publication No. WO 98/30679, for example.

A culture can be “xeno-Free (XF)” which refers to a medium or a culturecondition, which is essentially free from heterogeneous animal-derivedcomponents. For culturing human cells, any proteins of a non-humananimal, such as mouse, would be xeno components. Thus, in someembodiments, the disclosed conditions are xeno-free. Thus, for culturinghuman cells, a culture including human AB serum can be “xeno free.”

Other culturing conditions can be appropriately defined. For example,the culturing temperature can be about 30 to 40° C., for example, atleast or about 31, 32, 33, 34, 35, 36, 37, 38, 39° C. but particularlynot limited to them. In one embodiment, the cells are cultured at 37° C.The CO₂ concentration can be about 1 to 10%, for example, about 2% to5%, or any range derivable therein. In one non-limiting example, about5% CO₂ concentration is utilized.

The primary cultures are stimulated with an effective amount ofallogenic irradiated feeder cells and a cytokine, such as interleukin(IL)-15 or IL-2, to form stimulated T cells. The feeder cells can be,for example, allogeneic irradiated peripheral blood mononuclear cells.Feeder cells, including human feeder cells, can be irradiated, such aswith 4000 rad of gamma irradiation.

In some embodiment, the cells are also stimulated with a polyclonal Tcells stimulator, such as phytohemagglutinin. In some non-limitingexamples, a concentration of 1 μg/ml to 2 μg/ml is utilized. In furtherembodiments, both a cytokine, such as IL-15 and/or IL-2, and PHA areutilized.

In some embodiments, a ratio is used such that about 1,000 to about2,000 CD8⁺CD39⁺CD103⁺ T cells are stimulated with about 100,000 to about300,000 allogeneic feeder cells, such as irradiated allogeneic PBMC. Inother embodiments, a ratio is used such that about 1,000 to about 2,000CD8⁺CD39⁺CD103⁺ T cells are stimulated with about 200,000 allogeneicfeeder cells.

A cytokine, such as IL-2 or IL-15 can be included in the culture. Insome embodiments, IL-15 can be used at a concentration of about 5 ng/mlto about 15 ng/ml of IL-15. In some embodiments, IL-15 is included inthe culture at a concentration of about 7 ng/ml to about 12 ng/ml, suchas at concentration of about 7, about 8, about 9, about 10, about 11, orabout 12 ng/ml. In one non-limiting example, IL-15 is included at aconcentration of about 10 ng/ml.

The stimulated CD39+CD103+CD8+ T cell cultures are then replenished withfresh tissue culture medium and the cytokine, such as IL-15 and/or IL-2,throughout the in-vitro expansion. IL-15 can be included in this tissueculture medium at a concentration of about 5 ng/ml to about 50 ng/ml ofIL-15, such as about 10 ng/ml to about 50 ng/ml of IL-15. In someembodiments, IL-15 is included in the culture at a concentration ofabout 7 ng/ml to about 12 ng/ml, such as at concentration of about 7,about 8, about 9, about 10, about 11, or about 12 ng/ml. In onenon-limiting example, IL-15 is included at a concentration of about 10ng/ml. In other examples, IL-15 is included at a concentration of about20, about 30, about 40, or about 50 ng/ml.

The cell culture can be maintained for any period. In some embodiments,following 15 to 30 days in culture, the expanded CD39+CD103+CD8+ T cellsare harvested.

Methods of Isolating and Using a Nucleic Acid Encoding T Cell Receptors

Methods are provided for isolating nucleic acid encoding T cellreceptors (TCRs) that specifically bind tumor cell antigens. Thesemethods include isolating CD39⁺CD8⁺ T cells, such as CD39+CD103+CD8 Tcells, from a sample from a subject with a tumor expressing the tumorcell antigen. In some embodiments, the tumor is a solid tumor, such as ahead and neck squamous cell carcinoma, lung cancer, melanoma, ovariancancer renal cell carcinoma, bladder cancer, cervical cancer, livercancer, prostate cancer, breast cancer, glioblastoma or rectal cancer.The subject can be a human subject or a veterinary subject. The samplecan be any sample from the subject, including, but not limited to, aperipheral blood sample or a tumor biopsy. Methods for isolatingCD8⁺CD39⁺ T cells, such as CD39+CD103+CD8 T cells, are disclosed above.

In some embodiments, the methods include expanding the CD8⁺CD39⁺ Tcells, such as CD39+CD103+CD8 T cells. Methods expanding for theCD8⁺CD39⁺ T cells, such as CD39+CD103+CD8 T cells, in vitro are alsodisclosed above. In other embodiments, primary CD8⁺CD39⁺ T cells, suchas CD39+CD103+CD8 T cells, are utilized, wherein the cells are notexpanded in vitro.

The methods further include cloning a nucleic acid molecule encoding aTCR from the CD8⁺CD39⁺ T cells, such as CD39+CD103+CD8 T cells. Methodsfor cloning TCRs are known in the art, see for example, U.S. Pat. No.8,697,854, incorporated herein by reference. TCR's are members of theimmunoglobulin superfamily and usually consist of and α- and β-subunits.These possess one N-terminal immunoglobulin (Ig)-variable (V) domain,one Ig-constant (C) domain, a transmembrane/cell membrane-spanningregion, and a short cytoplasmic tail at the C-terminal end. The variabledomains of both the TCR α-chain and β-chain have three hypervariable orcomplementarity determining regions (CDRs), whereas the variable regionof the β-chain has an additional area of hypervariability (HV4) thatdoes not normally contact antigen and therefore is not considered a CDR.

CDR3 is the primary CDR that recognizes processed antigen, although CDR1of the alpha chain has also been shown to interact with the N-terminalpart of an antigenic peptide. CDR1 of the β-chain also interacts withthe C-terminal part of the peptide. CDR2 is thought to recognize theMHC. CDR4 of the β-chain is not thought to participate in antigenrecognition, but has been shown to interact with superantigens. Theconstant domain of the TCR domain consists of short connecting sequencesin which a cysteine residue forms disulfide bonds, which forms a linkbetween the two chains. The affinity of TCR's for a specific antigenmakes them of use therapeutically. For example, tumors can beeffectively treated by using adoptive immunotherapy with T cellsexpressing a specific TCR. Methods for cloning TCRs, and for usingadoptive immunotherapy using cells transfected with TCRs, are disclosedin PCT Publication No. WO 2006/031221, U.S. Pat. No. 5,906,936: PCTPublication No. WO97/32603; PCT Publication No. WO2007/065957, and PCTPublication No. WO2008/039818. Methods of generating nucleic acidmolecules encoding TCR and T cells (or NK cells) including suchreceptors are disclosed, for example, in Brentjens et al., 2010,Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, MolecularTherapy, published online Feb. 23, 2010, pages 1-9; Till et al., 2008,Blood, 1 12:2261-2271; Park et al., Trends Biotechnol., 29:550-557,2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J.Hematol Oncol., 6:47, 2013; PCT Pub. WO2012/079000, WO2013/126726; andU.S. Pub. 2012/0213783, each of which is incorporated by referenceherein in its entirety).

In some embodiments, single cell RNA sequences are used for the T cellreceptors or pair-seq (Adaptive). In one non-limiting example, purifiedT cells are isolated to single cells using a fluidigm or 10× genomicsinstrument. T Cell DNA is then amplified and sequenced. T cells do notneed to be primed or have antigen presented for this process.

Additional examples of appropriate cloning and sequencing techniques,and instructions sufficient to direct persons of skill through manycloning exercises are known (see, e.g, Sambrook et al. (MolecularCloning: A Laboratory Manual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012)and Ausubel et al. (In Current Protocols in Molecular Biology, JohnWiley & Sons, New York, through supplement 104, 2013). Productinformation from manufacturers of biological reagents and experimentalequipment also provide useful information. Such manufacturers includethe SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis,Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

Nucleic acid sequences encoding the TCR can be prepared by any suitablemethod. Once the entire sequence is cloned and known, it can also beprepared by direct chemical synthesis by methods such as thephosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979;the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151,1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862, 1981; the solid phase phosphoramidite triester methoddescribed by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981,for example, using an automated synthesizer as described in, forexample, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168,1984; and the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis produces a single stranded oligonucleotide. This can beconverted into double stranded DNA by hybridization with a complementarysequence or by polymerization with a DNA polymerase using the singlestrand as a template.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill. Modifications can bemade to a nucleic acid encoding a polypeptide described herein withoutdiminishing its biological activity. Some modifications can be made tofacilitate the cloning, expression, or incorporation of the targetingmolecule into a fusion protein. Such modifications are well known tothose of skill in the art and include, for example, termination codons,a methionine added at the amino terminus to provide an initiation, site,additional amino acids placed on either terminus to create convenientlylocated restriction sites, or additional amino acids (such as poly His)to aid in purification steps.

The nucleic acid molecule encoding the TCR can operably linked to aheterologous promoter. The nucleic acid molecule encoding the TCR can beincluded in a vector (such as a lentiviral vector or gamma retroviralvector) for expression in a host cell. Exemplary cells are mammaliancells, and include a T cell, such as a cytotoxic T lymphocyte (CTL) anda NK cell. In specific non-limiting examples, the cell is a T cell, suchas a CD3⁺ T cell. The CD3⁺ T cell can be a CD4⁺ or a CD8⁺ T cell.

The nucleic acid molecules also can be expressed in a recombinantlyengineered cell such as bacteria, plant, yeast, insect and mammaliancells. The TCR can be expressed as a fusion protein. Methods ofexpressing and purifying antibodies and antigen binding fragments areknown and further described herein (see, e.g., Al-Rubeai (ed), AntibodyExpression and Production, Springer Press, 2011). Those of skill in theart are knowledgeable in the numerous expression systems available forexpression of proteins including E. coli, other bacterial hosts, yeast,and various higher eukaryotic cells such as the COS, CHO, HeLa andmyeloma cell lines. The term “host cell” also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. Methods of stable transfer, meaning that the foreignDNA is continuously maintained in the host, are known in the art. Asdisclosed herein, specific embodiments of the present disclosure includeT cells, such as human T cells and human NK cells, which express theTCR. These T cells can be CD3⁺ T cells, such as CD4+ or CD8⁺ T cells.

The expression of nucleic acids encoding the TCR to a promoter (which iseither constitutive or inducible), followed by incorporation into anexpression cassette. The promoter can be any promoter of interest,including a cytomegalovirus promoter and a human T cell lymphotrophicvirus promoter (HTLV)-1. Optionally, an enhancer, such as acytomegalovirus enhancer, is included in the construct. The cassettescan be suitable for replication and integration in either prokaryotes oreukaryotes. Typical expression cassettes contain specific sequencesuseful for regulation of the expression of the DNA encoding the protein.For example, the expression cassettes can include appropriate promoters,enhancers, transcription and translation terminators, initiationsequences, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, sequences for the maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons. The vector can encode a selectable marker, such as amarker encoding drug resistance (for example, ampicillin or tetracyclineresistance).

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation (internal ribosomal binding sequences), and atranscription/translation terminator For eukaryotic cells, the controlsequences can include a promoter and/or an enhancer derived from, forexample, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and apolyadenylation sequence, and can further include splice donor and/oracceptor sequences (for example, CMV and/or HTLV splice acceptor anddonor sequences).

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Cells transformed by thecassettes can be selected by resistance to antibiotics conferred bygenes contained in the cassettes, such as the amp, gpt, neo and hyggenes.

Eukaryotic cells can also be cotransformed with polynucleotide sequencesencoding the TCR, and a second foreign DNA molecule encoding aselectable phenotype, such as the herpes simplex thymidine kinase gene.Another method is to use a eukaryotic viral vector, such as simian virus40 (SV40), a lentivirus or bovine papilloma virus, to transiently infector transform eukaryotic cells and express the protein (see for example,Viral Expression Vectors, Springer press, Muzyczka ed., 2011). One ofskill in the art can readily use an expression systems such as plasmidsand vectors of use in producing proteins in cells including highereukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

In some embodiments, a viral vector is utilized for expression of theTCR. Viral vectors include, but are not limited to simian virus 40,adenoviruses, adeno-associated virus (AAV), lentiviral vectors, andretroviruses, such as gamma retroviruses. Retroviral vectors provide ahighly efficient method for gene transfer into eukaryotic cells.Moreover, retroviral integration takes place in a controlled fashion andresults in the stable integration of one or a few copies of the newgenetic information per cell. Without being bound by theory, lentiviralvectors have the advantage over vectors derived from onco-retrovirusessuch as murine leukemia viruses in that they can transducenon-proliferating cells, such as hepatocytes. They also have the addedadvantage of low immunogenicity. The use of lentiviral vectors toexpress a TCR is known in the art, and is disclosed for example in U.S.Application No. 2014/0050708, which is incorporated herein by reference.A transposon can be used.

In some embodiments, host cells are produced for introduction intosubject of interest. The host cell can be a peripheral blood lymphocyte(PBL) or a peripheral blood mononuclear cell (PBMC), a purified T cell,or a purified NK cell. The T cell can be any T cell, such as a culturedT cell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal (such as ahuman patient to which the TCR-T cell will later be administered). Ifobtained from a mammalian subject, such as a human subject, the T cellcan be obtained from numerous sources, including but not limited toblood, bone marrow, lymph node, the thymus, or other tissues or fluids.T cells can also be enriched for or purified. The T cell can be any typeof T cell and can be of any developmental stage, including but notlimited to, CD3⁺ cells, CD4⁺/CD8⁺ double positive T cells, CD4⁺ helper Tcells, e.g., Th₁ and Th₂ cells, CD8⁺ T cells (e.g., cytotoxic T cells),tumor infiltrating cells, memory T cells, naive T cells, and the like.The T cell may be a CD3⁺ T cell, such as a CD8⁺ T cell or a CD4⁺ T cell.In alternative embodiments, the cell can be an NK cells, such as an NKcell obtained from the same subject to which the TCR-NK cell will laterbe administered. In some embodiments, the cells are human. In otherembodiments, the cells are from a veterinary subject.

Also provided is a population of cells comprising at least one host celldescribed herein. The population of cells can be a heterogeneouspopulation comprising the host cell comprising any of the recombinantexpression vectors encoding the TCR, in addition to at least one othercell, e.g., a host cell (e.g., a T cell), which does not comprise anyrecombinant expression vector, or a cell other than a T cell, e.g., a Bcell, a macrophage, a neutrophil, an erythrocyte, etc. Alternatively,the population of cells can be a substantially homogeneous population,in which the population comprises mainly host cells (e.g., consistingessentially of) comprising the recombinant expression vector encodingthe TCR. The population also can be a clonal population of cells, inwhich all cells of the population are clones of a single host cellcomprising a recombinant expression vector, such that all cells of thepopulation comprise the recombinant expression vector. In one embodimentof the invention, the population of cells is a clonal populationcomprising host cells comprising a recombinant expression vector asdescribed herein. The T cells can be CD3⁺ T cells, such as CD8+ T cellor a CD4⁺ T cells. The cells can also be NK cells.

The cells can be autologous to a recipient. The recipient can have atumor, or be at risk for having a tumor. The recipient can haveundergone prior treatment for a tumor. The tumor can be a solid tumor,such as solid tumor is a head and neck squamous cell carcinoma, lungcancer, melanoma, ovarian cancer renal cell carcinoma, bladder cancer,cervical cancer, liver cancer, prostate cancer, breast cancer,glioblastoma or rectal cancer. In some embodiments, autologous T cellsor NK cells are isolated from a subject, such as a human subject, andthe isolated TCR is introduced into these cells. These transformed cellsare then re-introduced to the subject. In this scenario, the donor andthe recipient are the same subject. The subject can be human. In someembodiments, a subject is administered a therapeutically effectiveamount of T cells and/or NK cells expressing the cloned TCR. Inparticular embodiments (see U.S. Published Application No. US20140271635A1, incorporated herein by reference), prior to expansion and geneticmodification, a source of T cells is obtained from a subject.

In some embodiments, the T and/or NK cells are autologous. In otherembodiments, the T cells and/or NK cells are allogeneic. The T cellsand/or NK cells are then introduced into the subject, as disclosedabove. In one embodiment, the cells transiently express the vector for4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. In onenon-limiting example, the vector is transduced into the T cell byelectroporation.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, dogs, cats, mice, rats, pigs (and other veterinarysubjects) and non-human primates. T cells can be obtained from a numberof sources, including peripheral blood mononuclear cells, bone marrow,lymph node tissue, cord blood, thymus tissue, tissue from a site ofinfection, ascites, pleural effusion, spleen tissue, and tumors. Inother embodiments, any number of T cell lines available in the art, maybe used. In some embodiments, subjects can undergo leukapheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., T cells and or NKcells. These cell isolates may be expanded by methods known in the artand treated such that a TCR construct is introduced, thereby creating anautologous cell that express the cloned TCR. In one aspect, TCRexpressing cells are generated using viral vector, such as, but notlimited to, lentiviral viral vectors. In some non-limiting examples, Tcells and/or NK cells can be obtained from a unit of blood collectedfrom a subject using any number of techniques known to the skilledartisan, such as FICOLL™ separation, or the cells can be obtained byapheresis. The apheresis product typically contains lymphocytes,including T cells, monocytes, granulocytes, B cells, NK cells, othernucleated white blood cells, red blood cells, and platelets.

Cells collected by apheresis can be washed to remove the plasma fractionand to place the cells in an appropriate buffer or media for subsequentprocessing steps. In some non-limiting examples, the cells are washedwith phosphate buffered saline (PBS). In an alternative examples, thewash solution lacks calcium and may lack magnesium or may lack many ifnot all divalent cations. Initial activation steps in the absence ofcalcium can lead to magnified activation. The washing step can beaccomplished by methods known in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CYTOMATE®, or the HAEMONETICS CELL SAVER 5@)according to the manufacturer's instructions. After washing, the cellscan be resuspended in a variety of biocompatible buffers, such as asaline solution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample can be removed and the cells directlyresuspended in culture media.

In some embodiments, T cells are isolated from peripheral bloodlymphocytes by negative selection. A specific subpopulation of T cells,such as CD3+, CD4+, CD8+, CD28+CD45RA+, and CD45RO+ T cells, naïveand/or memory T cells, can be further isolated by positive or negativeselection techniques. For example, T cells can isolated by incubationwith anti-CD3/anti-CD28 conjugated beads, such as DYNABEADS® M-450CD3/CD28 T, or CD3/IL-2, for a time period sufficient for positiveselection of the desired T cells, see for example U.S. PublishedApplication No. US20140271635 A1. In a non-limiting example the timeperiod is about 24 to about 72 hours and all integer values therebetween. In further non-limiting examples, the time period is at least24 hours, 36, 48 hours or longer. Longer incubation times can be used toisolate T cells in any situation where there are few T cells as comparedto other cell types, such in isolation from immunocompromisedindividuals. Multiple rounds of selection can also be used.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD8a, CD14, CD15, CD16, CD19,CD36, CD56, CD132, TCR γ/δ, and CD235a (Glycophorin A). To enrich forCD8+ cells by negative selection, the monoclonal antibody cocktailcontains antibodies to CD4, CD15, CD16, CD19, CD34, CD36, CD56, CD123,TCR γ/δ, and CD235a. A T cell population can be selected that expressesone or more cytokines. Methods for screening for cell expression aredisclosed in PCT Publication No. WO 2013/126712.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied to ensure maximum contact of cells and beads. Insome embodiments, a concentration of 1 million cells/ml is used. Infurther embodiments, greater than 100 million cells/ml is used. In otherembodiments, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,50, 65, 70, 75, 80, 85, 90, 95, or 100 million cells/ml is used. Withoutbeing bound by theory, using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Lower concentrations ofcells can also be used. Without being bound by theory, significantlydiluting the mixture of T cells and surface (e.g., particles such asbeads), interactions between the particles and cells is minimized. Thisselects for cells that express high amounts of desired antigens to bebound to the particles. In some embodiments, the concentration of cellsused is 5×10⁶/ml. In other embodiments, the concentration used can befrom about 1×10⁵/ml to 1×10⁶/ml, and any integer value in between.

Cells can be incubated on a rotator for varying lengths of time atvarying speeds at either 2-10° C. or at room temperature. T cells forstimulation can also be frozen after a washing step. Without being boundby theory, the freeze and subsequent thaw step provides a more uniformproduct by removing granulocytes and to some extent monocytes in thecell population. After the washing step that removes plasma andplatelets, the cells can be suspended in a freezing solution. While manyfreezing solutions and parameters are known in the art and will beuseful in this context, one method involves using PBS containing 20%DMSO and 8% human serum albumin, or culture media containing 10% Dextran40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25%Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5%Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cellfreezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen, see U.S. Publication No.US-2014-0271635 A1.

Blood samples or apheresis product can be collected from a subject at atime period prior to when the expanded cells as described herein mightbe needed. As such, the source of the cells to be expanded can becollected at any time point necessary, and desired cells, such as Tcells, isolated and frozen for later use in T cell therapy for anynumber of diseases or conditions that would benefit from T cell therapy,such as those described herein. In one aspect a blood sample or anapheresis is taken from a generally healthy subject. In certain aspects,a blood sample or an apheresis is taken from a generally healthy subjectwho is at risk of developing a disease, such as a tumor, but who has notyet developed a disease, and the cells of interest are isolated andfrozen for later use. In certain aspects, the T cells may be expanded,frozen, and used at a later time. In certain aspects, samples arecollected from a patient shortly after diagnosis of a particulardisease, such as a tumor, as described herein but prior to anytreatments. In a further aspect, the cells are isolated from a bloodsample or an apheresis from a subject prior to any number of relevanttreatment modalities. In certain aspects, cryopreserved cells are thawedand washed as described herein and allowed to rest for one hour at roomtemperature prior to use. Blood samples or apheresis product can becollected from a subject when needed, and not frozen. In someembodiments, autologous tumor-bearing T cells are isolated fromindividuals for subsequent transfection and infusion.

T cells can be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005. T cells can be expanded by contact with a surface havingattached thereto an agent that stimulates a CD3/TCR complex associatedsignal and a ligand that stimulates a costimulatory molecule on thesurface of the T cells. T cells can also be expanded using PHA or bulkPBMCs can to be transfected with CD3/28 or CD3/IL2. In some non-limitingexamples, T cell populations may be stimulated as described herein, suchas by contact with an anti-CD3 antibody, or antigen-binding fragmentthereof, or an anti-CD2 antibody immobilized on a surface, or by contactwith a protein kinase C activator (e.g., bryostatin) in conjunction witha calcium ionophore. For co-stimulation of an accessory molecule on thesurface of the T cells, a ligand that binds the accessory molecule isused. For example, a population of T cells can be contacted with ananti-CD3 antibody and an anti-CD28 antibody, under conditionsappropriate for stimulating proliferation of the T cells. To stimulateproliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibodyinclude 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used ascan other methods commonly known in the art (Berg et al., TransplantProc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol. Meth. 227(1-2):53-63,1999). Other methods include the use of allogeneic, irradiated PBMC'stogether with phytohaemagglutinin to stimulate T cell proliferation.

Once a TCR is isolated, various assays can be used to evaluate theactivity of the molecule, such as but not limited to, the ability toexpand T cells following antigen stimulation, sustain T cell expansionin the absence of re-stimulation, and anti-cancer activities inappropriate in vitro and animal models. In some embodiments, theupregluation of activation markers, such as, but not limited to, 1-1BBis evaluated, such as by flow cytometry.

The method includes administering to the subject a therapeuticallyeffective amount of the pharmaceutical composition cells, such as Tcells and/or NK cells, that express the cloned TCR. Subjects in needthereof, such as a subject with a tumor, may subsequently undergostandard treatment with chemotherapy or surgery (for cancer) oranti-viral agents (for an HIV infection). The administration of the hostcells expressing the heterologous TCR can result in treating the tumor,such as decreasing tumor volume, decreasing metastasis, or decreasing asign or symptom of the tumor.

Pharmaceutical compositions can include a TCR-expressing host cell,e.g., a plurality of TCR-expressing host cells, as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. The TCR-expressing hostcells can be T cells, such as CD3⁺ T cells, such as CD4⁺ and/or CD8⁺ Tcells, and/or NK cells. Such compositions may include buffers such asneutral buffered saline, phosphate buffered saline and the like;carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol;proteins; polypeptides or amino acids such as glycine; antioxidants;chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives.

With regard to the cells, a variety of aqueous carriers can be used, forexample, buffered saline and the like, for introducing the cells. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the subject's needs.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, such asendotoxin, mycoplasma, replication competent lentivirus (RCL), p24,VSV-G nucleic acid, HIV gag, residual anti-CD8/anti-CD39/anti-CD103coated beads, mouse antibodies, pooled human serum, bovine serumalbumin, bovine serum, culture media components, vector packaging cellor plasmid components, a bacterium and a fungus.

The precise amount of the composition to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of metastasis, and condition of thepatient (subject). It can generally be stated that a pharmaceuticalcomposition comprising the T cells (and/or NK cells) described hereinmay be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, suchas 10⁵ to 10⁶ cells/kg body weight, including all integer values withinthose ranges. Exemplary doses are 10⁶ cells/kg to about 1×10⁸ cells/kg,such as from about 5×10⁶ cells/kg to about 7.5×10⁷ cells/kg, such as atabout 2.5×10⁷ cells/kg, or at about 5.0×10⁷ cells/kg.

A composition can be administered once or multiple times, such as 2, 3,4, 5, 6, 7, 8, 9, or 10 times at these dosages. The composition can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The compositions can be administered daily, weekly,bimonthly or monthly. In some non-limiting examples, the composition isformulated for intravenous administration and is administered multipletimes. The quantity and frequency of administration will be determinedby such factors as the condition of the subject, and the type andseverity of the subject's disease, although appropriate dosages may bedetermined by clinical trials.

In one embodiment, the TCR is introduced into cells, such T cells or NKcells, and the subject receives an initial administration of cells, andone or more subsequent administrations of the cells, wherein the one ormore subsequent administrations are administered less than 15 days,e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after theprevious administration. In one embodiment, more than one administrationof the cells is provided to the subject (e.g., human) per week, e.g., 2,3, or 4 administrations of the TCR expressing cells are administered perweek. In one embodiment, the subject receives more than oneadministration of the T cells per week (e.g., 2, 3 or 4 administrationsper week) (also referred to as a cycle), followed by a week of noadministrations, and then one or more additional administration of theTCR expressing cells (e.g., more than one administration of the cellsper week) is administered to the subject. In another embodiment, thesubject (e.g., human subject) receives more than one cycle of TCRexpressing cells, and the time between each cycle is less than 10, 9, 8,7, 6, 5, 4, or 3 days. In one embodiment, the TCR expressing cells areadministered every other day for 3 administrations per week. In anotherembodiment, the TCR expressing cells are administered for at least two,three, four, five, six, seven, eight or more weeks. The dosage of theabove treatments to be administered to a patient will vary with theprecise nature of the condition being treated and the recipient of thetreatment. The scaling of dosages for human administration can beperformed according to art-accepted practices.

In some embodiments, TCR-modified T cells are able to replicate in vivoresulting in long-term persistence that can lead to sustained tumorcontrol. In various aspects, the T cells administered to the subject, orthe progeny of these cells, persist in the subject for at least fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, eleven months, twelve months, thirteen months,fourteen month, fifteen months, sixteen months, seventeen months,eighteen months, nineteen months, twenty months, twenty-one months,twenty-two months, twenty-three months, or for years afteradministration of the T cell to the subject. In other embodiments, thecells and their progeny are present for less than six months, fivemonth, four months, three months two months, or one month, e.g., threeweeks, two weeks, one week, after administration of the T cell to thesubject.

The administration of the subject compositions may be carried out in anyconvenient manner, including by injection, transfusion, implantation ortransplantation. The disclosed compositions can be administered to apatient trans arterially, subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous (i.v.)injection, or intraperitoneally. In some embodiments, the compositionsare administered to a patient by intradermal or subcutaneous injection.In other embodiments, the compositions of the present invention areadministered by i.v. injection. The compositions can also be injecteddirectly into a tumor or lymph node.

In one embodiment, compositions containing isolated populations of cellscan also contain one or more additional pharmaceutical agents, such asone or more anti-microbial agents (for example, antibiotics, anti-viralagents and anti-fungal agents), anti-tumor agents (for example,fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide,doxorubicin, or vincristine), depleting agents (for example,fludarabine, etoposide, doxorubicin, or vincristine), or non-steroidalanti-inflammatory agents such as acetylsalicylic acid, ibuprofen ornaproxen sodium), cytokines (for example, interleukin-2), or a vaccine.Many chemotherapeutic agents are presently known in the art. In oneembodiment, the chemotherapeutic agent is selected from the groupconsisting of mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, anti-survival agents,biological response modifiers, anti-hormones, e.g. anti-androgens, andanti-angiogenesis agents.

For the treatment of malignancy, the method can also includeadministering to the subject a therapeutically effective amount of anadditional cancer therapeutic (including chemotherapeutic agents andradiation) or surgery.

The cells expressing the TCR can be administered in conjunction withsurgery, radiation, chemotherapy, or immunotherapy. Suitablechemotherapeutic agents are disclosed above. Cells expressing the TCRcan also be administered with a PD-1, CTLA-4, TIM-3, LAG3, BTLAAntagonist and/or a 4-1BB Agonist, as disclosed in detail below.

Methods of Detecting and Treatment

It is disclosed herein that administration of CD39⁺CD8⁺ T cells, such asCD39+CD103+CD8 T cells, are of use in diagnosis and treatment. In theseembodiments, CD39⁺CD8⁺ T cells, such as CD39+CD103+CD8 T cells, aremeasured in a biological sample from a subject. In some embodiments, thesample is a peripheral blood sample or a tumor biopsy. The subject canbe any subject, such as a human or a veterinary subject. In furtherembodiments, the subject has a tumor, is suspected of having a tumor, oris at risk of having a tumor. The tumor can be a solid tumor. In somenon-limiting examples, the solid tumor is a head and neck squamous cellcarcinoma, lung cancer, melanoma, ovarian cancer renal cell carcinoma,bladder cancer, cervical cancer, liver cancer, prostate cancer, breastcancer, glioblastoma or rectal cancer.

In some embodiments, methods are disclosed for determining if a subjectwith a tumor will respond to a cancer therapeutic, which include, but anot limited to, biological response modifiers (such as cytokines andchemokines), cancer vaccines, chemotherapeutic agents, immunotherapeuticagents, and radiation. In some embodiments, the cancer therapeutic canbe a checkpoint inhibitor, a 4-1BB agonist and/or radiation. The cancertherapeutic can be a chemical. The methods can also be used to detect ifa subject will respond to surgery.

These methods include detecting the presence of CD39⁺CD8⁺ T cells, suchas CD39+CD103+CD8 T cells, in a biological sample from a subject,wherein the presence of the CD39+CD8⁺ T cells, such as CD39+CD103+CD8 Tcells, in the biological sample that the cancer therapeutic, such as,but not limited, to, a checkpoint inhibitor, 4-1BB agonist, and/orradiation, will be effective for treating the tumor in the subject. Themethod can also indicate that the subject will respond to a surgicalprocedure, such as resection. The method can also include administeringthe cancer therapeutic to the subject, or performing the surgicalprocedure. In some non-limiting examples, the cancer therapeutic is acheckpoint inhibitor, which can be, without limitation, a PD-1antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist,a TIM-3 antagonist or a LAG3 antagonist. Suitable antagonists aredisclosed in detail below. Suitable 4-1BB agonists are also disclosedbelow.

In other embodiments, methods are disclosed for determining if a subjectwith a tumor will respond to a therapeutic regimen, such as including acancer therapeutic. The cancer therapeutic can be a chemotherapeuticagent or radiation. The cancer therapeutic can be a checkpointinhibitor, such as a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4antagonist, a BTLA antagonist, a TIM-3 antagonist or a LAG3 antagonist,or can be a 4-1BB agonist. In some embodiments, the methods includeadministering to a subject a first dose of the cancer therapeutic, anddetermining the number of CD39⁺CD8⁺ T cells, such as CD39+CD103+CD8 Tcells, in a biological sample from a subject. An increase in the amountof CD39⁺CD8⁺ T cells, such as CD39+CD103+CD8 T cells, in the biologicalsample as compared to a control indicates that the first dose of thecancer therapeutic is effective for treating the tumor in the subject.

In further embodiments, methods are disclosed for determining if asubject with a tumor will respond to a cancer therapeutic. The cancertherapeutic can be a chemotherapeutic agent or radiation. The cancertherapeutic can be a checkpoint inhibitor, such as a PD-1 antagonist, aPD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3antagonist or a LAG3 antagonist, or can be a 4-1BB agonist. Thesemethods include administering to a subject a first dose of the cancertherapeutic, and determining the number of CD39+CD8+ T cells, such asCD39+CD103+CD8 T cells, in a biological sample from a subject, whereinan increase in the amount of CD39+CD8+ T cells, such as CD39+CD103+CD8 Tcells, in the biological sample as compared to a control indicates thatthe first dose of the cancer therapeutic is effective for treating thetumor in the subject. In additional embodiments, the methods furtherinclude administering a second dose of the cancer therapeutic to thesubject, wherein the first dose is the same as the second dose, orwherein the second dose is lower than the first dose.

In yet other embodiments, methods are disclosed for treating a subjectwith a tumor. These methods include administering to a subject a firstdose of the cancer therapeutic, and determining the number of CD39+CD8+T cells, such as CD39+CD103+CD8 T cells, in a biological sample from asubject. A decrease or no change in the amount of CD39+CD8+ T cells,such as CD39+CD103+CD8 T cells, in the biological sample as compared toa control indicates that the first dose of the cancer therapeutic is noteffective for treating the tumor in the subject. A second dose of thecancer therapeutic is administered to the subject, wherein the seconddose is higher than the first dose, or wherein the second dose is thesame as the first dose.

In some embodiments, the subject has tumor, or is at risk of developinga tumor, as discussed above. These subjects can be identified bystandard methods suitable by one of skill in the art, such as aphysician. The disclosed methods include selecting a subject ofinterest, and administering the cancer therapeutic of interest,including, but not limited to a checkpoint inhibitor, such as a PD-1antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist,a TIM-3 antagonist or a LAG3 antagonist. The subject can also beadministered a 4-1BB agonist. The method can detect subjects that willrespond to a surgical procedure.

In additional embodiments, the subject is administered a therapeuticallyeffective amount of CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells,and a therapeutically effective amount of a cancer therapeutic, such as,but not limited to, a checkpoint inhibitor, for example a PD-1antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist,a TIM-3 antagonist or a LAG3 antagonist. In additional embodiments,subject can be administered a 4-1BB agonist. Administration of thepurified CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, and acheckpoint inhibitor or a 4-1BB agonist, as disclosed herein, willincrease the ability of a subject to overcome pathological conditions,such as a tumor. The cells and the checkpoint inhibitor can be includedin a single pharmaceutical composition or in separate pharmaceuticalcompositions. Therefore, by purifying and generating a purifiedpopulation of CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, from asubject ex vivo and introducing a therapeutic amount of these cells, theimmune response of the recipient subject is enhanced. The administrationof a therapeutically effective amount of a checkpoint inhibitor or a4-1BB agonist also enhances the immune response of the recipient. Thus,the methods disclosed herein for determining if a chemotherapeutic agentor radiation is effective can be used in combination with any of thetherapeutic methods (and in any of the subjects) described above.

The methods can also be used to evaluate the dose of a cancertherapeutic that is therapeutically effective for a subject. Forexample, the methods disclosed herein can be used to determine if thedose administered to a subject of interest can be lowered and still beeffective. The methods disclosed herein also can be used to determine ifthe dose administered to a subject is too low, and thus must beincreased to be therapeutically effective.

Any of the disclosed methods can include measuring other cell types,such as B and/or T cells. The disclosed methods can also includemeasuring the expression of markers such as PD-1, PD-L1, CTLA-4, BTLA,TIM-3 or LAG3. The expression of CD8, CD39 and/or CD103 is evaluated.

In some embodiments, CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells,are measured. An increase in the number of CD39+CD8+ T cells, such asCD39+CD103+CD8 T cells, from the biological sample as compared to acontrol indicates that the dose of the cancer therapeutic is of usetreating the subject, and wherein an absence of a significant alterationin the number of CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, ascompared to the control indicates that the dose of the cancertherapeutic is not of use to treat the subject. The control can be apreviously determined standard value, or the quantity of CD39+CD8+ Tcells, such as CD39+CD103+CD8 T cells, in a sample from the subjectprior to the administration of the cancer therapeutic, or the quantityof CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, in a sample fromthe subject, when the subject tis administered a control substance.

Generally, measuring the number of CD39+CD8+ T cells, such asCD39+CD103+CD8 T cells, includes obtaining a sample that includes Tcells from a subject, and determining the presence or number ofCD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, in the sample. Insome examples, the sample is a biopsy sample, a blood sample, or asample of peripheral blood mononuclear cells. The methods includeimmunohistochemistry and/or flow cytometry.

The methods can include immunohistochemistry methods, such as on abiological sample from a subject. The sample can be a tumor sample. Insome embodiments, an antibody (or antigen binding fragment), such as anantibody that binds CD8, CD39 or CD103 is directly labeled with adetectable label. In another embodiment, the antibody (or antigenbinding fragment) that binds CD8, CD39 or CD103 (the first antibody) isunlabeled and a second antibody or other molecule that can bind theantibody that binds the first antibody is utilized. As is well known toone of skill in the art, a second antibody is chosen that is able tospecifically bind the specific species and class of the first antibody.For example, if the first antibody is a human IgG, then the secondaryantibody may be an anti-human-IgG. Other molecules that can bind toantibodies include, without limitation, Protein A and Protein G, both ofwhich are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Cells can also be quantitated using flow cytometry. In additionalembodiments, the sample can be purified, for example to separate Tcells, such as CD8 T cells or CD39+CD8+ T cells, such as CD39+CD103+CD8T cells. In some embodiments, the methods include measuring the quantityof CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells. In some examples,the quantity of CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, in abiological sample is compared to a control. Suitable controls are notedabove.

In some examples, cell suspensions are produced from a tumor sample. Inone non-limiting example, under sterile conditions, tumors are cut intosmall pieces and digested in RPMI-1640 with hyaluronidase, collagenase,DNase as well as human serum albumin. Cells can be digested, forexample, for 1 hour at room temperature under agitation with a magneticstir bar. Cell suspensions are filtered through a cell filter. Tumorinfiltrating lymphocytes can be enriched as by centrifugation with adensity gradient solution.

Methods for isolating, detecting, and/or quantitating T cells are knownin the art, and exemplary protocols are provided herein. Methods alsoare known in the art to measure the proliferation of T cells. Thesemethods generally involve the use of molecular and/or biochemicaltechniques and not simple visual observation. Cells in some examples,fluorescence activated cell analyses (FACS) is utilized. FACS can beused to sort (isolate) cells such as T cells by staining the cells withan appropriately labeled antibody. In one embodiment, several antibodies(such as antibodies that bind CD8, CD39 and CD103) and FACS sorting canbe used to produce substantially purified populations of CD39+CD8+ Tcells, such as CD39+CD103+CD8 T cells. Any FACS technique can beemployed, see, for example, methods of FACS disclosed in U.S. Pat. No.5,061,620.

However, other techniques of differing efficacy can be employed topurify and isolate desired populations of cells. The separationtechniques employed should maximize the retention of viability of thefraction of the cells to be collected. The particular technique employedwill, of course, depend upon the efficiency of separation, cytotoxicityof the method, the ease and speed of separation, and what equipmentand/or technical skill is required.

Separation procedures include magnetic separation, using antibody-coatedmagnetic beads, cytotoxic agents, either joined to a monoclonal antibodyor used in conjunction with complement, and “panning,” which utilizes amonoclonal antibody attached to a solid matrix, or another convenienttechnique. Antibodies attached to magnetic beads and other solidmatrices, such as agarose beads, polystyrene beads, hollow fibermembranes and plastic petri dishes, allow for direct separation. Cellsthat are bound by the antibody can be removed from the cell suspensionby simply physically separating the solid support from the cellsuspension. The exact conditions and duration of incubation of the cellswith the solid phase-linked antibodies will depend upon several factorsspecific to the system employed. The selection of appropriateconditions, however, is well within the skill in the art.

The unbound cells then can be eluted or washed away with physiologicbuffer after sufficient time has been allowed for the cells expressing amarker of interest (e.g., CD39 or CD103) to bind to the solid-phaselinked antibodies. The bound cells are then separated from the solidphase by any appropriate method, depending mainly upon the nature of thesolid phase and the antibody employed.

Antibodies can be conjugated to biotin, which then can be removed withavidin or streptavidin bound to a support, or fluorochromes, which canbe used, with FACS, to enable cell separation.

For example, cells expressing CD8 or CD3 are initially separated fromother cells by the cell-surface expression of CD8 or CD3. Purity of theisolated CD8⁺ cells or CD3⁺ cells is then checked, such as with a BDLSRFORTESSA® flow cytometer (Becton Dickinson, San Jose, Calif.), if sodesired. In one embodiment, further purification steps are performed,such as FACS sorting the population of cells. In one example, thissorting can be performed to detect expression of CD39, CD103, and CD8.

The methods can also include measuring cell proliferation. Methods foranalyzing cell proliferation, such as the assessment of theproliferation are known in the art. For example, membrane dye dilutionapproaches can be utilized which include ex vivo chemical labeling ofcells of interest with fluorescent dyes. Labeling with tritiatednucleoside analogues (commonly ³H-thymidine deoxyribonucleoside, ³H-TdR)or bromodeoxyuridine (BrdU) can be utilized. FACS analysis is availablefor the measurement of BrdU incorporation. Surrogate markers ofproliferation such as DNA content and cell cycle-associated proteins,can also be used.

In one example, measurement of Ki67 or PCNA can be utilized. Ki67antigen is the prototypic cell cycle related nuclear protein that isexpressed by proliferating cells in all phases of the active cell cycle(G1, S, G2 and M phase). It is absent in resting (G0) cells. Ki67antibodies are useful in establishing proliferation. Ki67 antibodies canbe used to quantify proliferating cells among and resting cells (Ki67index). Ki67 is routinely used as a marker of cell cycling andproliferation; antibodies to Ki67 are commercially available, such asfrom ABCAM®, and methods are available to use these antibodies inimmunohistochemical and FACS analyses.

Other methods can be used to detect those cells that are in the activecell cycle at the time of sampling. Proliferation of lymphocytes, suchas CD39+CD8+ T cells, for example CD39+CD103+CD8 T cells, can also bemeasured by using methods that utilize stable isotopes to label DNA inbiological samples including cells. DNA is uniformly and highly labeledvia the de novo synthesis pathway. The stable isotope labels used, e.g.²H-glucose or heavy water (²H₂O or H₂ ¹⁸O), are non-toxic to animals andhumans, and generally regarded as safe by the US Food and DrugAdministration (FDA) (see U.S. Patent Application Publication No.2009/0155179). The measurement of stable isotope label incorporationinto lymphocyte DNA comprises the following steps: (i) extraction of DNAor its release from chromatin without further isolation, hydrolysis ofDNA to deoxyribonucleotides, (ii) selective release of deoxyribose frompurine deoxyribonucleotides, (iii) derivatization of purine deoxyriboseto a volatile derivative (e.g., pentane tetraacetate, pentafluorobenzyltetraacetyl derivative, or another suitable derivative) suitable foranalysis by gas chromatography/mass spectrometry (GC/MS), (iv) GC/MSanalysis of said derivative, (v) analysis of the pattern of massisotopomer abundance of said derivative, and (vi) calculation from saidpattern of an excess enrichment value that is a measure of stableisotope incorporation. Specific embodiments of each of these methodshave been taught (see U.S. Pat. No. 5,910,40).

PD-1, CTLA-4, TIM-3, LAG3, BTLA Antagonists and 4-1BB Agonists

Check-point inhibitors, such as PD-1 antagonists, PD-L1 antagonists,CTLA-4 antagonists, LAG3 antagonists, TIM-3 antagonists and/or BTLAantagonists are of use in the method disclosed herein, for example incombination with CD8⁺CD39⁺CD103⁺ T cells. 4-1BB agonists are also of usein the method disclosed herein. The PD-1 antagonist, PD-L1 antagonist,CTLA-4 antagonist, LAG3 antagonist, TIM-3 antagonist, BTLA antagonist,and/or 4-1BB agonist can be a chemical or biological compound. The agentcan be an antibody, including but not limited to a chimeric, humanized,or human antibody. Suitable antagonists and agonists also includeantigen binding fragments of these antibodies (see above for adescription of antigen binding fragments). The antagonist can be, forexample, an inhibitor nucleic acid molecule or a small molecule, such asa molecule less than 900 daltons or less than 800 daltons.

A PD-1 antagonist can be any chemical compound or biological moleculethat blocks binding of PD-L1 or PD-L2 expressed on a cell to human PD-1expressed on an immune cell (T cell, B cell or NKT cell). Alternativenames or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279and SLEB2 for PD-1; PDCD1L1, PD-L1, B7H1, B7-4, CD274 and B7-H forPD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. Exemplaryhuman PD-1 amino acid sequences can be found in NCBI Accession No.:NP_005009. Exemplary human PD-L1 and PD-L2 amino acid sequences can befound in NCBI Accession No.: NP_054862 and NP_079515, respectively, Apr.28, 2017, incorporated by reference. In vivo, PD-1 is expressed onactivated T cells, B cells, and monocytes. In humans, PD-1 is a 50-55kDa type I transmembrane receptor that was originally identified in a Tcell line undergoing activation-induced apoptosis. PD-1 is expressed onT cells, B cells, and macrophages. The ligands for PD-1 are the B7family members PD-ligand 1 (PD-L1, also known as B7-H1) and PD-L2 (alsoknown as B7-DC). A PD-L1 or PD-L2 inhibitor can be used in the methodsdisclosed herein.

Experimental data implicates the interactions of PD-1 with its ligandsin downregulation of central and peripheral immune responses. Inparticular, proliferation in wild-type T cells but not in PD-1-deficientT cells is inhibited in the presence of PD-L1. (See e.g., Ishida et al.,EMBO J. 11:3887, 1992; Shinohara et al. Genomics 23:704, 1994; U.S. Pat.No. 5,698,520, incorporated herein by reference).

Additional PD-1 amino acid sequences are disclosed in U.S. Pat. No.6,808,710 and U.S. Patent Application Publication Nos. 2004/0137577,2003/0232323, 2003/0166531, 2003/0064380, 2003/0044768, 2003/0039653,2002/0164600, 2002/0160000, 2002/0110836, 2002/0107363, and2002/0106730, which are incorporated herein by reference.

PD-1 is a member of the CD28/CTLA-4 family of molecules based on itsability to bind to PD-L1. In vivo, like CTLA-4, PD-1 is rapidly inducedon the surface of T-cells in response to anti-CD3 (Agata et al. Int.Immunol. 8:765, 1996). T cell exhaustion is concomitant with aninduction in PD-1 expression, see PCT Publication No. 2008/083174,incorporated herein by reference. T-cell cytotoxicity can be increasedby contacting a T-cell with an agent that reduces the expression oractivity of PD-1. An agent that reduces the expression or activity ofPD-1 can be used to increase an immune response, such as to a tumor.Without being bound by theory, reduction of PD-1 expression or activityresults in an increase in cytotoxic T cell activity, increasing thespecific immune response.

PD-1 family members bind to one or more receptors, such as PD-L1 andPD-L2 on antigen presenting cells. An exemplary amino acid sequence forPD-L1 is provided as GENBANK® Accession No. AAG18508, which isincorporated by reference herein as available Oct. 4, 2000. An exemplaryPD-L2 precursor amino acid sequence is provided as GENBANK® AccessionNo. AAK15370, which is incorporated by reference herein as availableApr. 8, 2002. An exemplary variant PD-L2 precursor amino acid sequenceis provided as GENBANK® Accession No. Q9BQ51, which is incorporated byreference herein as available Dec. 12, 2006.

Antagonists of use in the methods disclosed herein include agents thatreduce the expression or activity of a PD ligand 1 (PD-L1) or a PDligand 2 (PD-L2) or reduces the interaction between PD-1 and PD-L1 orthe interaction between PD-1 and PD-L2; these are PD-antagonists.Exemplary compounds include antibodies (such as an anti-PD-1 antibody,an anti-PD-L1 antibody, and an anti-PD-L2 antibody), RNAi molecules(such as anti-PD-1 RNAi molecules, anti-PD-L1 RNAi, and an anti-PD-L2RNAi), antisense molecules (such as an anti-PD-1 antisense RNA, ananti-PD-L1 antisense RNA, and an anti-PD-L2 antisense RNA), dominantnegative proteins (such as a dominant negative PD-1 protein, a dominantnegative PD-L1 protein, and a dominant negative PD-L2 protein), andsmall molecule inhibitors. Any of these PD-1 antagonists are of use inthe methods disclosed herein.

Other antibodies are of use in the methods disclosed herein (such as ananti-CTLA-4 antibody, and anti-LAG3 antibody, an-anti-TIM-3 antibody oran anti-BTLA antibody), RNAi molecules (such as anti-CTLA-4 RNAimolecules, anti-LAG3 RNAi, anti-TIM-3 RNAi and an anti-BTLA RNAi),antisense molecules (such as an anti-CTLA-4 antisense RNA, anti-LAG3antisense RNA, anti-TIM-3 antisense RNA and an anti-BTLA antisense RNA).Dominant negative proteins also of use are a dominant negative CTLA-4protein, a dominant negative LAG3 protein, a dominant negative LAG-3protein and a dominant negative BTLA protein). Any of these antagonistsare of use in the methods disclosed herein. In addition, 4-1BB agonists,such as antibodies that bind 4-1BB and RNA Aptamers, are of use in themethods disclosed herein. A TGF-β receptor inhibitory or dominantnegative protein is also of use.

An antagonist is an agent having the ability to reduce the expression orthe activity of the target in a cell. In some embodiments, PD-1, PD-L1,PD-L2, LAG3, TIM-3, CTLA-4 or BTLA expression or activity is reduced byat least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%compared to such expression or activity in a control. Exemplaryreductions in activity are at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, or a complete absence of detectable activity. In one example, thecontrol is a cell that has not been treated with the PD-1 antagonist. Inanother example, the control is a standard value, or a cell contactedwith an agent, such as a carrier, known not to affect activity.Expression or activity can be determined by any standard method in theart. In one non-limiting example, a PD-1 antagonist inhibits or reducesbinding of PD-1 to PD-L1, PD-L2, or both. In one non-limiting example, aPD-L1 antagonist reduces the binding of PD-L1 or PD-1.

An agonist is an agent having the ability to increase the expression orthe activity of the target in a cell. In some embodiments, 4-1BBexpression or activity is increased by at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to such expression oractivity in a control. Exemplary increases in activity are at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, or a complete absence ofdetectable activity. In one example, the control is a cell that has notbeen treated with the 4-1BB agonist. In another example, the control isa standard value, or a cell contacted with an agent, such as a carrier,known not to affect activity. Expression or activity can be determinedby any standard method in the art. In one non-limiting example, 4-1BBagonist stimulates or increases binding.

A. Antibodies

In some embodiments, the antagonist is an antibody. Exemplary amino acidsequence of antibodies that bind PD-1 are disclosed, for example, inU.S. Patent Publication No. 2006/0210567, which is incorporated hereinby reference. Antibodies that bind PD-1 are also disclosed in U.S.Patent Publication No. 2006/0034826, which is also incorporated hereinby reference. Antibodies that bind PD-1 are also disclosed in U.S. Pat.Nos. 7,488,802, 7,521,051, 8,008,449, 8,354,509, 8,168,757, and U.S. PCTPublication No. WO2004/004771, PCT Publication No. WO2004/072286, PCTPublication No. WO2004/056875, and US Published Patent Application No.2011/0271358. The antibody can be KEYTRUDA® (pembrolizumab). Theantibody can be an anti-PD-1 antibody such as Nivolumab(ONO-4538/BMS-936558) or OPDIVO® from Ono Pharmaceuticals. PD-L1 bindingantagonists include YW243.55. S70, MPDL3280A, MDX-1105 and MEDI 4736,see U.S. Published Patent Application No. 2017/0044256. Examples ofmonoclonal antibodies that specifically bind to human PD-L1, and areuseful in the disclosed methods and compositions are disclosed in PCTPublication No. WO2013/019906, PCT Publication No. WO2010/077634 A1 andU.S. Pat. No. 8,383,796. The checkpoint inhibitor antibodies againstPD-1 (e.g., Nivolumab, pidilizumab, and Pembrolizumab) or PD-L1 (e.g.,Durvalumab, Atezolizumab, and Avelumab) are of use in any of the methodsdisclosed herein. Antibodies that bind PD-1, PD-L2 and PD-1 are alsodisclosed in U.S. Pat. No. 8,552,154. In several examples, the antibodyspecifically binds CTLA-4, BTLA, PD-1, PD-L1, or PD-L2 with an affinityconstant of at least 10⁷ M⁻¹, such as at least 10⁸ M⁻¹ at least 5×10⁸M⁻¹ or at least 10⁹ M⁻¹. Any of these antibodies, and antigen bindingfragments, are of use in the methods disclosed herein.

Exemplary antibodies that specifically bind CTLA-4 are disclosed in PCTPublication No. WO 2001/014424, PCT Publication No. WO 2004/035607, U.S.Publication No. 2005/0201994, European Patent No. EP1141028, andEuropean Patent No. EP 1212422 B1. Additional CTLA-4 antibodies aredisclosed in U.S. Pat. Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720,6,682,736, 6,207,156, 5,977,318, 6,682,736, 7,109,003, 7,132,281,7,452,535, and 7,605,238; PCT Publication No. WO 01/14424, PCTPublication No. WO 00/37504, PCT Publication No. WO 98/42752, U.S.Published Patent Application No. 2000/037504, U.S. Published ApplicationNo. 2002/0039581, and U.S. Published Application No. 2002/086014.Antibodies that specifically bind CTLA-4 are also disclosed in Hurwitzet al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camachoet al., J. Clin. Oncol., 22(145): Abstract No. 2505 (2004) (antibodyCP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998). In someembodiments the CTLA-4 antagonist is Ipilmumab (also known as MDX-010and MDX-101 and YERVOY®), see PCT Publication No. WO 2001/014424,incorporated herein by reference. These antibodies, and antigen bindingfragments, are of use in the methods disclosed herein.

In further embodiments, a BTLA antagonist is utilized in the methodsdisclosed herein. Antibodies that specifically bind BTLA are disclosed,for example, in U.S. Published Patent Application No. 2016/0222114, U.S.Published Patent Application No. 2015/0147344, and U.S. published PatentApplication No. 2012/0288500, all incorporated herein by reference.Biological agents that modulate BTLA activity, specifically usingHerpesvirus entry mediator (HVEM) cis complexes are disclosed in U.S.Published Patent Application No. 2014/0220051 and U.S. Published PatentApplication No. 2010/0104559, both incorporated herein by reference. Inyet other embodiments, the antibody specifically binds TIM-3, such asTSR-022. In further embodiments, the antibody specifically binds LAG3,such as BMS-986016, GSK2831781, or the antibodies disclosed in PCTPublication No. WO2015042246 A1, incorporated herein by reference. Seealso Clinical trial number NCT01968109 for “Safety Study of Anti-LAG-3With and Without Anti-PD-1 in the Treatment of Solid Tumors” availableon the internet at clinicaltrials.gov and incorporated by referenceherein. These antibodies, and antigen binding fragments, are of use inthe methods disclosed herein.

A 4-1BB agonist antibody is also of use. Suitable antibodies aredisclosed, for example, in U.S. Pat. No. 8,337,850 and PCT PublicationNo. WO 2015179236 A1, both incorporated by reference herein. Antibodiesof use in any of the disclosed methods include urelumab (BMS-663513) andPF-05082566 (Pfizer).

The antibodies of use in the disclosed methods include monoclonalantibodies, humanized antibodies, deimmunized antibodies (such as toreduce a human-anti-mouse response), chimeric antibodies, andimmunoglobulin (Ig) fusion proteins. Antigen binding fragments of theseantibodies are also of use in the methods disclosed herein. Polyclonalantibodies can be prepared by one of skill in the art, such as byimmunizing a suitable subject (such as a veterinary subject) with animmunogen. The antibody titer in the immunized subject can be monitoredover time by standard techniques, such as with an enzyme linkedimmunosorbent assay (ELISA) using immobilized antigen. In one example,an antibody that specifically bind CTLA-4, BTLA, TIM-3, LAG3, PD-1,PD-L1, or PD-L2 (or combinations thereof) can be isolated from themammal (such as from serum) and further purified by techniques known toone of skill in the art. For example, antibodies can be purified usingprotein A chromatography to isolate IgG antibodies.

Antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques (see Kohler andMilstein Nature 256:495 49, 1995; Brown et al., J. Immunol. 127:539 46,1981; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77 96, 1985; Gefter, M. L. et al. (1977) Somatic CellGenet. 3:231 36; Kenneth, R. H. in Monoclonal Antibodies: A NewDimension In Biological Analyses. Plenum Publishing Corp., New York,N.Y. (1980); Kozbor et al. Immunol. Today 4:72, 1983; Lerner, E. A.(1981) Yale J. Biol. Med. 54:387 402; Yeh et al., Proc. Natl. Acad. Sci.76:2927 31, 1976). In one example, an immortal cell line (typically amyeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with PD-1, PD-L1, PD-L2, TIM-3, LAG3, BTLA or CTLA-4 and theculture supernatants of the resulting hybridoma cells are screened toidentify a hybridoma producing a monoclonal antibody that specificallybinds to the polypeptide of interest.

In one embodiment, to produce a hybridoma, an immortal cell line (suchas a myeloma cell line) is derived from the same mammalian species asthe lymphocytes. For example, murine hybridomas can be made by fusinglymphocytes from a mouse immunized with a CTLA-4, BTLA, TIM-3, LAG3,PD-1, PD-L1, PD-L2, or 4-1BB peptide with an immortalized mouse cellline. In one example, a mouse myeloma cell line is utilized that issensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, including,for example, P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines,which are available from the American Type Culture Collection (ATCC),Rockville, Md. HAT-sensitive mouse myeloma cells can be fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfused(and unproductively fused) myeloma cells. Hybridoma cells producing amonoclonal antibody of interest can be detected, for example, byscreening the hybridoma culture supernatants for the productionantibodies that bind a PD-1, PD-L1, TIM-3, LAG3, BTLA, CTLA-4, PD-L2 or4-1BB molecule, such as by using an immunological assay (such as anenzyme-linked immunosorbant assay(ELISA) or radioimmunoassay (RIA).

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal antibody that specifically binds CTLA-4, BTLA, TIM-3, LAG3,PD-1, PD-L1, PD-L2 or 4-1BB can be identified and isolated by screeninga recombinant combinatorial immunoglobulin library (such as an antibodyphage display library) with CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1,PD-L2 or 4-1BB to isolate immunoglobulin library members thatspecifically bind the polypeptide. Kits for generating and screeningphage display libraries are commercially available (such as, but notlimited to, Pharmacia and Stratagene). Examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication No. WO 90/02809; PCT Publication No. WO 91/17271; PCTPublication No. WO 92/18619; PCT Publication WO 92/20791; PCTPublication No. WO 92/15679; PCT Publication No. WO 92/01047; PCTPublication WO 93/01288; PCT Publication No. WO 92/09690; Barbas et al.,Proc. Natl. Acad. Sci. USA 88:7978 7982, 1991; Hoogenboom et al.,Nucleic Acids Res. 19:4133 4137, 1991.

In one example the sequence of the specificity determining regions ofeach CDR is determined. Residues are outside the SDR (non-ligandcontacting sites) are substituted. For example, in any of the CDRsequences as in the table above, at most one, two or three amino acidscan be substituted. The production of chimeric antibodies, which includea framework region from one antibody and the CDRs from a differentantibody, is well known in the art. For example, humanized antibodiescan be routinely produced. The antibody or antibody fragment can be ahumanized immunoglobulin having complementarity determining regions(CDRs) from a donor monoclonal antibody that binds CTLA-4, BTLA, TIM-3,LAG3, PD-1, PD-L1, PD-L2, or 4-1BB and immunoglobulin and heavy andlight chain variable region frameworks from human acceptorimmunoglobulin heavy and light chain frameworks. Humanized monoclonalantibodies can be produced by transferring donor complementaritydetermining regions (CDRs) from heavy and light variable chains of thedonor mouse immunoglobulin (such a CTLA-4, BTLA, TIM-3, LAG3, PD-1,PD-L1, PD-L2 or 4-1BB specific antibody) into a human variable domain,and then substituting human residues in the framework regions whenrequired to retain affinity. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith the immunogenicity of the constant regions of the donor antibody.Techniques for producing humanized monoclonal antibodies are described,for example, by Jones et al., Nature 321:522, 1986; Riechmann et al.,Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carteret al., Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev.Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993.The antibody may be of any isotype, but in several embodiments theantibody is an IgG, including but not limited to, IgG₁, IgG₂, IgG₃ andIgG₄. In some embodiments, the humanized immunoglobulin specificallybinds to the antigen of interest (e.g., CTLA-4, BTLA, TIM-3, LAG3, PD-1,PD-L1, PD-L2, or 4-1BB) with an affinity constant of at least 10⁷ M⁻¹,such as at least 10⁸ M⁻¹ at least 5×10⁸ M⁻¹ or at least 10⁹ M⁻¹.

In one embodiment, the sequence of the humanized immunoglobulin heavychain variable region framework can be at least about 65% identical tothe sequence of the donor immunoglobulin heavy chain variable regionframework. Thus, the sequence of the humanized immunoglobulin heavychain variable region framework can be at least about 75%, at leastabout 85%, at least about 99% or at least about 95%, identical to thesequence of the donor immunoglobulin heavy chain variable regionframework. Human framework regions, and mutations that can be made inhumanized antibody framework regions, are known in the art (see, forexample, in U.S. Pat. No. 5,585,089, which is incorporated herein byreference).

Antibodies, such as murine monoclonal antibodies, chimeric antibodies,and humanized antibodies, include full length molecules as well asfragments thereof, such as Fab, F(ab′)₂, and Fv which include a heavychain and light chain variable region and are capable of bindingspecific epitope determinants. These antibody fragments retain someability to selectively bind with their antigen or receptor. Thesefragments include:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule, can be        produced by digestion of whole antibody with the enzyme papain        to yield an intact light chain and a portion of one heavy chain;    -   (2) Fab′, the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain;        two Fab′ fragments are obtained per antibody molecule;    -   (3) (Fab′)₂, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)₂ is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   (4) Fv, a genetically engineered fragment containing the        variable region of the light chain and the variable region of        the heavy chain expressed as two chains; and    -   (5) Single chain antibody (such as scFv), defined as a        genetically engineered molecule containing the variable region        of the light chain, the variable region of the heavy chain,        linked by a suitable polypeptide linker as a genetically fused        single chain molecule.

Methods of making these antigen binding fragments are known in the art(see for example, Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1988). In several examples, thevariable region includes the variable region of the light chain and thevariable region of the heavy chain expressed as individual polypeptides.Fv antibodies are typically about 25 kDa and contain a completeantigen-binding site with three CDRs per each heavy chain and each lightchain. To produce these antibodies, the V_(H) and the V_(L) can beexpressed from two individual nucleic acid constructs in a host cell. Ifthe V_(H) and the V_(L) are expressed non-contiguously, the chains ofthe Fv antibody are typically held together by noncovalent interactions.However, these chains tend to dissociate upon dilution, so methods havebeen developed to crosslink the chains through glutaraldehyde,intermolecular disulfides, or a peptide linker. Thus, in one example,the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chainvariable region and the light chain variable region are chemicallylinked by disulfide bonds.

In an additional example, the Fv fragments comprise V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991;Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack etal., Bio/Technology 11:1271, 1993; and Sandhu, supra).

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. Nos. 4,036,945 and 4,331,647,and references contained therein; Nisonhoff et al., Arch. Biochem.Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al.,Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; andColigan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retainamino acid residues necessary for correct folding and stabilizingbetween the V_(H) and the V_(L) regions, and will retain the chargecharacteristics of the residues in order to preserve the low pI and lowtoxicity of the molecules. Amino acid substitutions (such as at mostone, at most two, at most three, at most four, or at most five aminoacid substitutions) can be made in the V_(H) and the V_(L) regions toincrease yield. Thus, one of skill in the art can readily review theamino acid sequence of an antibody of interest, locate one or more ofthe amino acids in the brief table above, identify a conservativesubstitution, and produce the conservative variant using well-knownmolecular techniques.

Effector molecules, such as therapeutic, diagnostic, or detectionmoieties can be linked to an antibody that specifically binds CTLA-4,BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB using any number ofmeans known to those of skill in the art. Both covalent and noncovalentattachment means may be used. The procedure for attaching an effectormolecule to an antibody varies according to the chemical structure ofthe effector. Polypeptides typically contain a variety of functionalgroups; such as carboxylic acid (COOH), free amine (—NH₂) or sulfhydryl(—SH) groups, which are available for reaction with a suitablefunctional group on an antibody to result in the binding of the effectormolecule. Alternatively, the antibody is derivatized to expose or attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, Ill. The linker can beany molecule used to join the antibody to the effector molecule. Thelinker is capable of forming covalent bonds to both the antibody and tothe effector molecule. Suitable linkers are well known to those of skillin the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the antibody and the effector molecule are polypeptides,the linkers may be joined to the constituent amino acids through theirside groups (such as through a disulfide linkage to cysteine) or to thealpha carbon amino and carboxyl groups of the terminal amino acids.

Nucleic acid sequences encoding the antibodies can be prepared by anysuitable method including, for example, cloning of appropriate sequencesor by direct chemical synthesis by methods such as the phosphotriestermethod of Narang et al., Meth. Enzymol. 68:90-99, 1979; thephosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979;the diethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862, 1981; the solid phase phosphoramidite triester methoddescribed by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981,for example, using an automated synthesizer as described in, forexample, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168,1984; and, the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis produces a single stranded oligonucleotide. This can beconverted into double stranded DNA by hybridization with a complementarysequence, or by polymerization with a DNA polymerase using the singlestrand as a template. One of skill would recognize that while chemicalsynthesis of DNA is generally limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Exemplary nucleic acids encoding sequences encoding an antibody thatspecifically binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or4-1BB can be prepared by cloning techniques. Examples of appropriatecloning and sequencing techniques, and instructions sufficient to directpersons of skill through many cloning exercises are found in Sambrook etal., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Productinformation from manufacturers of biological reagents and experimentalequipment also provide useful information. Such manufacturers includethe SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis,Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen (San Diego, Calif.), andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

In one example, an antibody of use is prepared by inserting the cDNAwhich encodes a variable region from an antibody that specifically bindsCTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB into a vectorwhich comprises the cDNA encoding an effector molecule (EM). Theinsertion is made so that the variable region and the EM are read inframe so that one continuous polypeptide is produced. Thus, the encodedpolypeptide contains a functional Fv region and a functional EM region.In one embodiment, cDNA encoding a detectable marker (such as an enzyme)is ligated to a scFv so that the marker is located at the carboxylterminus of the scFv. In another example, a detectable marker is locatedat the amino terminus of the scFv. In a further example, cDNA encoding adetectable marker is ligated to a heavy chain variable region of anantibody that specifically binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1,PD-L2, or 4-1BB so that the marker is located at the carboxyl terminusof the heavy chain variable region. The heavy chain-variable region cansubsequently be ligated to a light chain variable region of the antibodythat specifically binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2,or 4-1BB using disulfide bonds. In a yet another example, cDNA encodinga marker is ligated to a light chain variable region of an antibody thatbinds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB, so thatthe marker is located at the carboxyl terminus of the light chainvariable region. The light chain-variable region can subsequently beligated to a heavy chain variable region of the antibody thatspecifically binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or4-1BB using disulfide bonds.

Once the nucleic acids encoding the antibody or functional fragmentthereof are isolated and cloned, the protein can be expressed in arecombinantly engineered cell such as bacteria, plant, yeast, insect andmammalian cells. One or more DNA sequences encoding the antibody orfunctional fragment thereof can be expressed in vitro by DNA transferinto a suitable host cell. The cell may be prokaryotic or eukaryotic.The term also includes any progeny of the subject host cell. It isunderstood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. Methods ofstable transfer, meaning that the foreign DNA is continuously maintainedin the host, are known in the art.

Polynucleotide sequences encoding the antibody or functional fragmentthereof can be operatively linked to expression control sequences. Anexpression control sequence operatively linked to a coding sequence isligated such that expression of the coding sequence is achieved underconditions compatible with the expression control sequences. Theexpression control sequences include, but are not limited to appropriatepromoters, enhancers, transcription terminators, a start codon (i.e.,ATG) in front of a protein-encoding gene, splicing signal for introns,maintenance of the correct reading frame of that gene to permit propertranslation of mRNA, and stop codons.

The polynucleotide sequences encoding the antibody or functionalfragment thereof can be inserted into an expression vector including,but not limited to a plasmid, virus or other vehicle that can bemanipulated to allow insertion or incorporation of sequences and can beexpressed in either prokaryotes or eukaryotes. Hosts can includemicrobial, yeast, insect and mammalian organisms. Methods of expressingDNA sequences having eukaryotic or viral sequences in prokaryotes arewell known in the art. Biologically functional viral and plasmid DNAvectors capable of expression and replication in a host are known in theart.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody offunctional fragment thereof and a second foreign DNA molecule encoding aselectable phenotype, such as the herpes simplex thymidine kinase gene.Another method is to use a eukaryotic viral vector, such as simian virus40 (SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). One ofskill in the art can readily use expression systems such as plasmids andvectors of use in producing proteins in cells including highereukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide can becarried out by conventional means including preparative chromatographyand immunological separations. Once expressed, the recombinantantibodies can be purified according to standard procedures of the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y., 1982). Substantially purecompositions of at least about 90 to 95% homogeneity are disclosedherein, and 98 to 99% or more homogeneity can be used for pharmaceuticalpurposes. Once purified, partially or to homogeneity as desired, if tobe used therapeutically, the polypeptides should be substantially freeof endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991;Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544,1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970,incorporated by reference herein, and especially as described by Buchneret al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. It isdesirable to add excess oxidized glutathione or other oxidizing lowmolecular weight compounds to the refolding solution after theredox-shuffling is completed.

In addition to recombinant methods, the antibodies and functionalfragments thereof that are disclosed herein can also be constructed inwhole or in part using standard peptide synthesis. Solid phase synthesisof the polypeptides of less than about 50 amino acids in length can beaccomplished by attaching the C-terminal amino acid of the sequence toan insoluble support followed by sequential addition of the remainingamino acids in the sequence. Techniques for solid phase synthesis aredescribed by Barany & Merrifield, The Peptides: Analysis, Synthesis,Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp.3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, andStewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagent N,N′-dicycylohexylcarbodimide) are well known in the art.

B. Inhibitory Nucleic Acids

Inhibitory nucleic acids that decrease the expression and/or activity ofCTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 can also be used in themethods disclosed herein. One embodiment is a small inhibitory RNA(siRNA) for interference or inhibition of expression of a target gene.Nucleic acid sequences encoding PD-1, PD-L1 and PD-L2 are disclosed inGENBANK® Accession Nos. NM_005018, AF344424, NP_079515, and NP_054862,all incorporated by reference as available on Apr. 28, 2017.

Generally, siRNAs are generated by the cleavage of relatively longdouble-stranded RNA molecules by Dicer or DCL enzymes (Zamore, Science,296:1265-1269, 2002; Bernstein et al., Nature, 409:363-366, 2001). Inanimals and plants, siRNAs are assembled into RISC and guide thesequence specific ribonucleolytic activity of RISC, thereby resulting inthe cleavage of mRNAs or other RNA target molecules in the cytoplasm. Inthe nucleus, siRNAs also guide heterochromatin-associated histone andDNA methylation, resulting in transcriptional silencing of individualgenes or large chromatin domains. PD-1 siRNAs are commerciallyavailable, such as from Santa Cruz Biotechnology, Inc.

The present disclosure provides RNA suitable for interference orinhibition of expression of a target gene, which RNA includes doublestranded RNA of about 15 to about 40 nucleotides containing a 0 to5-nucleotide 3′ and/or 5′ overhang on each strand. The sequence of theRNA is substantially identical to a portion of an mRNA or transcript ofa target gene, such as CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2,for which interference or inhibition of expression is desired. Forpurposes of this disclosure, a sequence of the RNA “substantiallyidentical” to a specific portion of the mRNA or transcript of the targetgene for which interference or inhibition of expression is desireddiffers by no more than about 30 percent, and in some embodiments nomore than about 10 percent, from the specific portion of the mRNA ortranscript of the target gene. In particular embodiments, the sequenceof the RNA is exactly identical to a specific portion of the mRNA ortranscript of the target gene.

Thus, siRNAs disclosed herein include double-stranded RNA of about 15 toabout 40 nucleotides in length and a 3′ or 5′ overhang having a lengthof 0 to 5-nucleotides on each strand, wherein the sequence of the doublestranded RNA is substantially identical to (see above) a portion of amRNA or transcript of a nucleic acid encoding CTLA-4, BTLA, TIM-3, LAG3,PD-1, PD-L1, or PD-L2. In particular examples, the double stranded RNAcontains about 19 to about 25 nucleotides, for instance 20, 21, or 22nucleotides substantially identical to a nucleic acid encoding CTLA-4,BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2. In additional examples, thedouble stranded RNA contains about 19 to about 25 nucleotides 100%identical to a nucleic acid encoding CTLA-4, BTLA, TIM-3, LAG3, PD-1,PD-L1, or PD-L2. It should be not that in this context “about” refers tointeger amounts only. In one example, “about” 20 nucleotides refers to anucleotide of 19 to 21 nucleotides in length.

Regarding the overhang on the double-stranded RNA, the length of theoverhang is independent between the two strands, in that the length ofone overhang is not dependent on the length of the overhang on otherstrand. In specific examples, the length of the 3′ or 5′ overhang is0-nucleotide on at least one strand, and in some cases it is0-nucleotide on both strands (thus, a blunt dsRNA). In other examples,the length of the 3′ or 5′ overhang is 1-nucleotide to 5-nucleotides onat least one strand. More particularly, in some examples the length ofthe 3′ or 5′ overhang is 2-nucleotides on at least one strand, or2-nucleotides on both strands. In particular examples, the dsRNAmolecule has 3′ overhangs of 2-nucleotides on both strands.

Thus, in one particular provided RNA embodiment, the double-stranded RNAcontains 20, 21, or 22 nucleotides, and the length of the 3′ overhang is2-nucleotides on both strands. In embodiments of the RNAs providedherein, the double-stranded RNA contains about 40-60% adenine+uracil(AU) and about 60-40% guanine+cytosine (GC). More particularly, inspecific examples the double-stranded RNA contains about 50% AU andabout 50% GC.

Also described herein are RNAs that further include at least onemodified ribonucleotide, for instance in the sense strand of thedouble-stranded RNA. In particular examples, the modified ribonucleotideis in the 3′ overhang of at least one strand, or more particularly inthe 3′ overhang of the sense strand. It is particularly contemplatedthat examples of modified ribonucleotides include ribonucleotides thatinclude a detectable label (for instance, a fluorophore, such asrhodamine or FITC), a thiophosphate nucleotide analog, a deoxynucleotide(considered modified because the base molecule is ribonucleic acid), a2′-fluorouracil, a 2′-aminouracil, a 2′-aminocytidine, a 4-thiouracil, a5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, an inosine, ora 2′O-Me-nucleotide analog.

Antisense and ribozyme molecules for CTLA-4, BTLA, TIM-3, LAG3, PD-1,PD-L1, or PD-L2 are also of use in the method disclosed herein.Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate an mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target cell producing CTLA-4, BTLA,TIM-3, LAG3, PD-1, PD-L1, or PD-L2. The use of antisense methods toinhibit the in vitro translation of genes is well known in the art (see,for example, Marcus-Sakura, Anal. Biochem. 172:289, 1988).

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleicacid can be constructed using chemical synthesis and enzymatic ligationreactions using procedures known in the art. For example, an antisensenucleic acid molecule can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, such as phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridin-e,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, amongst others.

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the bloomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., AntisenseRes. and Dev. 1(3):227, 1991; Helene, C., Anticancer Drug Design6(6):569), 1991. This type of inhibitory oligonucleotide is also of usein the methods disclosed herein.

Ribozymes, which are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases, are also of use. Through the modificationof nucleotide sequences which encode these RNAs, it is possible toengineer molecules that recognize specific nucleotide sequences in anRNA molecule and cleave it (Cech, J. Amer. Med. Assn. 260:3030, 1988). Amajor advantage of this approach is that, because they aresequence-specific, only mRNAs with particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-base recognition sequences are preferable to shorter recognitionsequences.

Various delivery systems are known and can be used to administer thesiRNAs and other inhibitory nucleic acid molecules as therapeutics. Suchsystems include, for example, encapsulation in liposomes,microparticles, microcapsules, nanoparticles, recombinant cells capableof expressing the therapeutic molecule(s) (see, e.g., Wu et al., J.Biol. Chem. 262, 4429, 1987), construction of a therapeutic nucleic acidas part of a retroviral or other vector, and the like.

C. Small Molecules

CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 antagonists, and 4-1BBagonist, include molecules that are identified from large libraries ofboth natural product or synthetic (or semi-synthetic) extracts orchemical libraries according to methods known in the art. The screeningmethods that detect decreases in CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1,or PD-L2 activity (such as detecting cell death for PD-1, PD-L1 andPD-L2) are useful for identifying compounds from a variety of sourcesfor activity. Screening methods that detect increases in 4-1BB activityare also of use for identifying compounds from such sources. The initialscreens may be performed using a diverse library of compounds, a varietyof other compounds and compound libraries. Thus, molecules that bindCTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 molecules that inhibitthe expression of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 andmolecules that inhibit the activity of CTLA-4, BTLA, TIM-3, LAG3, PD-1,PD-L1, or PD-L2 can be identified. Molecules that increase theexpression and/or activity of 4-1BB can also be identified. These smallmolecules can be identified from combinatorial libraries, naturalproduct libraries, or other small molecule libraries. In addition,CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, and PD-L2 antagonists, and 4-1BBagonists, can be identified as compounds from commercial sources, aswell as commercially available analogs of identified inhibitors. In someembodiments, the small molecule is less than 900 daltons, or less than800 daltons.

The precise source of test extracts or compounds is not critical to theidentification of antagonists. Accordingly, antagonists can beidentified from virtually any number of chemical extracts or compounds.Examples of such extracts or compounds that can be antagonists include,but are not limited to, plant-, fungal-, prokaryotic- or animal-basedextracts, fermentation broths, and synthetic compounds, as well asmodification of existing compounds. Numerous methods are also availablefor generating random or directed synthesis (e.g., semi-synthesis ortotal synthesis) of any number of chemical compounds, including, but notlimited to, saccharide-, lipid-, peptide-, and nucleic acid-basedcompounds. Synthetic compound libraries are commercially available fromBrandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee,Wis.). Agonists and antagonists can be identified from syntheticcompound libraries that are commercially available from a number ofcompanies including Maybridge Chemical Co. (Trevillet, Cornwall, UK),Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), andMicrosource (New Milford, Conn.). CTLA-4, BTLA, TIM-3, LAG3, PD-1,PD-L1, and PD-L2 antagonists, or 4-1BB agonists, can be identified froma rare chemical library, such as the library that is available fromAldrich (Milwaukee, Wis.). CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, andPD-L2 antagonists, or 4-1BB agonists, can be identified in libraries ofnatural compounds in the form of bacterial, fungal, plant, and animalextracts are commercially available from a number of sources, includingBiotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch OceangraphicsInstitute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.).Natural and synthetically produced libraries and compounds are readilymodified through conventional chemical, physical, and biochemical means.

Useful compounds may be found within numerous chemical classes, thoughtypically they are organic compounds, including small organic compounds.Small organic compounds have a molecular weight of more than 50 yet lessthan about 2,500 daltons, such as less than about 750 or less than about350 daltons can be utilized in the methods disclosed herein. Exemplaryclasses include heterocycles, peptides, saccharides, steroids, and thelike. The compounds may be modified to enhance efficacy, stability,pharmaceutical compatibility, and the like. In several embodiments,compounds of use has a Kd for CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, orPD-L2 of less than 1 nM, less than 10 nm, less than 1 μM, less than 10μM, or less than 1 mM.

D. Peptide Variants

An immunoadhesin that specifically binds to human CTLA-4, human BTLA,human TIM-3, human LAG3, human PD-1, human PD-L1, or human PD-L2 canalso be utilized. An immunoadhesin is a fusion protein containing theextracellular or a binding portion of a protein fused to a constantregion such as an Fc region of an immunoglobulin molecule. Examples ofimmunoadhesion molecules that specifically bind to PD-1 are disclosed inPCT Publication Nos. WO2010/027827 and WO2011/066342, both incorporatedby reference. These immunoadhesion molecules include AMP-224 (also knownas B7-DCIg), which is a PD-L2-FC fusion protein. Additional PD-1antagonists that are fusion proteins are disclosed, for example, in U.S.Published Patent Application No. 2014/0227262, incorporated herein byreference.

In one embodiment, a LAG3 antagonist of use in the disclosed methods isIMP321, a soluble LAG3, which has been used to activate dendritic cells.In another embodiment, aTIM-3 antagonists if use in the disclosedmethods is CA-327 (Curis).

A CTLA-4 antagonist can be a dominant negative protein or animmunoadhesins, see for example U.S. Published Patent Application No.2016/0264643, incorporated herein by reference. Additional anti-CTLA-4antagonists include any inhibitor, including but not limited to a smallmolecule, that can inhibit the ability of CTLA-4 to bind to its cognateligand, disrupt the ability of B7 to CTLA-4, disrupt the ability of CD80to bind to CTLA-4, disrupt the ability of CD86 to bind to CTLA-4.

In one embodiment, variants of a CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1,or PD-L2 protein which function as an antagonist can be identified byscreening combinatorial libraries of mutants, such as point mutants ortruncation mutants, of a CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, orPD-L2 protein to identify proteins with antagonist activity. In oneexample, the antagonist is a soluble protein.

In further embodiments, variants of 4-1BB that function as an agonistcan be identified by screening combinatorial libraries of 4-1BB mutants,such as point mutation or truncation mutants, to identify a protein withagonist activity. The agonist can be a soluble protein.

Thus, a library of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or4-1BB variants can be generated by combinatorial mutagenesis at thenucleic acid level and is encoded by a variegated gene library. Alibrary of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2 or 4-1BBvariants can be produced by, for example, by enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins (suchas for phage display) containing the set of sequences of interest.

There are a variety of methods, which can be used to produce librariesof potential CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2 or 4-1BBvariants from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be performed in an automatic DNAsynthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2 or 4-1BBsequences. Methods for synthesizing degenerate oligonucleotides areknown in the art (see, for example, Narang, et al., Tetrahedron 39:3,1983; Itakura et al. Annu. Rev. Biochem. 53:323, 1984; Itakura et al.Science 198:1056, 1984).

In addition, libraries of fragments of a CTLA-4, BTLA, TIM-3, LAG3,PD-1, PD-L1, PD-L2 or 4-1BB protein coding sequence can be used togenerate a population of fragments for screening and subsequentselection of variants of a specified antagonist (or agonist, in the caseof 4-1BB). In one embodiment, a library of coding sequence fragments canbe generated by treating a double stranded PCR fragment of a CTLA-4,BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of CTLA-4, BTLA, TIM-3, LAG3,PD-1, PD-L1, PD-L2, or 4-1BB.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of proteins. The most widelyused techniques, which are amenable to high through-put analysis, forscreening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM) can be usedin combination with the screening assays to identify CTLA-4, BTLA,TIM-3, LAG3, PD-1, PD-L1, or PD-L2 antagonists, or a 4-1BB agonist(Arkin and Youvan, Proc. Natl. Acad. Sci. USA 89:7811 7815, 1992;Delagrave et al., Protein Eng. 6(3):327 331, 1993).

In one embodiment, cell based assays can be exploited to analyze alibrary of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 variants.For example, a library of expression vectors can be transfected into acell line, which ordinarily synthesizes and secretes CTLA-4, BTLA,TIM-3, LAG3, PD-1, PD-L1, or PD-L2. The transfected cells are thencultured such that CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 anda particular CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2(respectively) variant are secreted. The effect of expression of themutant on activity in cells or in supernatants can be detected, such asby any of a functional assay. Plasmid DNA can then be recovered from thecells wherein endogenous activity is inhibited, and the individualclones further characterized.

Peptidomimetics can also be used as CTLA-4, BTLA, TIM-3, LAG3, PD-1,PD-L1, or PD-L2 antagonists. Peptide analogs are commonly used in thepharmaceutical industry as non-peptide drugs with properties analogousto those of the template peptide. These types of non-peptide compoundsand are usually developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar totherapeutically useful peptides can be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (for example, polypeptidethat has a PD-1 biological activity), but has one or more peptidelinkages optionally replaced by a —CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH.═.CH-(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO-linkages.These peptide linkages can be replaced by methods known in the art (see,for example, Morley, Trends Pharm. Sci. pp. 463 468, 1980; Hudson et al.Int. J. Pept. Prot. Res. 14:177 185, 1979; Spatola, Life Sci. 38:12431249, 1986; Holladay, et al. Tetrahedron Lett. 24:4401 4404, 1983).Peptide mimetics can be procured economical, be stable, and can haveincreased have-life or absorption. Labeling of peptidomimetics usuallyinvolves covalent attachment of one or more labels, directly or througha spacer (such as by an amide group), to non-interfering position(s) onthe peptidomimetic that are predicted by quantitative structure-activitydata and/or molecular modeling. Such non-interfering positions generallyare positions that do not form direct contacts with themacromolecules(s) to which the peptidomimetic binds to produce thetherapeutic effect. Derivitization of peptidomimetics should notsubstantially interfere with the desired biological or pharmacologicalactivity of the peptidomimetic.

A dominant negative protein or a nucleic acid encoding a dominantnegative protein that interferes with the biological activity of CTLA-4,BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 can also be used in the methodsdisclosed herein. A dominant negative protein is any amino acid moleculehaving a sequence that has at least 50%, 70%, 80%, 90%, 95%, or even 99%sequence identity to at least 10, 20, 35, 50, 100, or more than 150amino acids of the wild type protein to which the dominant negativeprotein corresponds. For example, a dominant-negative PD-L1 has mutationsuch that it binds PD-1 more tightly than native (wild-type) PD-1 butdoes not activate any cellular signaling through PD-1.

The dominant negative protein may be administered as an expressionvector. The expression vector may be a non-viral vector or a viralvector (e.g., retrovirus, recombinant adeno-associated virus, or arecombinant adenoviral vector). Alternatively, the dominant negativeprotein may be directly administered as a recombinant proteinsystemically or to the infected area using, for example, microinjectiontechniques.

Polypeptide antagonists can be produced in prokaryotic or eukaryotichost cells by expression of polynucleotides encoding the amino acidsequence, frequently as part of a larger polypeptide (a fusion protein,such as with ras or an enzyme). Alternatively, such peptides can besynthesized by chemical methods. Methods for expression of heterologousproteins in recombinant hosts, chemical synthesis of polypeptides, andin vitro translation are well known in the art (see Maniatis el al.Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold SpringHarbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume 152,Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., SanDiego, Calif.; Kaiser et al., Science 243:187, 1989; Merrifield, Science232:342, 1986; Kent, Annu. Rev. Biochem. 57:957, 1988).

Peptides can be produced, such as by direct chemical synthesis, and usedas antagonists. Peptides can be produced as modified peptides, withnonpeptide moieties attached by covalent linkage to the N-terminusand/or C-terminus. In certain preferred embodiments, either thecarboxy-terminus or the amino-terminus, or both, are chemicallymodified. The most common modifications of the terminal amino andcarboxyl groups are acetylation and amidation, respectively.Amino-terminal modifications such as acylation (for example,acetylation) or alkylation (for example, methylation) andcarboxy-terminal-modifications such as amidation, as well as otherterminal modifications, including cyclization, can be incorporated intovarious embodiments. Certain amino-terminal and/or carboxy-terminalmodifications and/or peptide extensions to the core sequence can provideadvantageous physical, chemical, biochemical, and pharmacologicalproperties, such as: enhanced stability, increased potency and/orefficacy, resistance to serum proteases, desirable pharmacokineticproperties, and others.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Materials and Methods

Healthy donor blood samples and patient blood and tissue samples:Peripheral blood, uninvolved lymph nodes, metastatic lymph nodes andtumor samples were obtained from individuals with HNSCC, melanoma, coloncancer, rectal cancer, lung cancer, colorectal liver metastasis andovarian cancer. All subjects signed written informed consent approved bythe institute's Institutional Review Board (Providence Portland MedicalCenter, IRB).

At the time of sample collection, patients were not undergoing therapy.Previously, they had undergone a wide range of therapies, includingchemotherapy, radiotherapy, surgery and immunotherapy, or none of theabove.

Peripheral blood mononuclear cells were purified from whole blood overFicoll-Paque PLUS (GE Healthcare) gradient and cryopreserved prior toanalysis.

Tumor specimens were prepared as follows: Briefly, under sterileconditions, tumors were cut into small pieces and digested in RPMI-1640supplemented with Hyaluronidase at 0.5 mg/ml, Collagenase at 1 mg/ml(both Sigma-Aldrich), DNase at 30 U/ml (Roche) as well as human serumalbumin (MP Biomedicals) at 1.5% final concentration. Cells weredigested for 1 hr at room temperature under agitation with a magneticstir bar. Cell suspensions were filtered through a 70 μm filter. Tumorinfiltrating lymphocytes were enriched as described above byFicoll-Paque PLUS density centrifugation. Tumor single-cell suspensionswere cryopreserved until further analysis.

Antibodies and flow cytometry: Fluorescently labeled antibodies werepurchased from the following manufacturers:

Biolegend: CD3 (UCHT1), CD4 (OKT-4 or RPA-T4), CD8 (RPA-T8), CD25(BC96), CD38 (HIT2), CD45RA (HI100), CD69 (FN50), HLA-DR (L243), CTLA-4(BNI3), 4-1BB (4B4-1), CCR7 (G043H7), Granzyme B (GB11), IFN-g (4S.B3),TNF-a (Mab11)

BD Bioscience: CD27 (M-T271), CD127 (HIL-7R-M21), PD-1 (EH12), Ki-67(B56),

eBioscience: CD28 (CD28.2), CD39 (eBioA1), CD103 (Ber-ACT8 and B-Ly7),FOXP3 (PCH101), ICOS (ISA-3)

R&D: TIM-3 (344823)

A fixable live/dead dye was used to distinguish viable cells(Biolegend). Cell surface staining was performed in FACS buffer (PBS,supplemented with 1% FBS and 0.01% NaN₃). Intracellular staining wasperformed using the Fix/Perm kit from eBioscience according to themanufacturer's instructions. To analyze cytokine production byperipheral blood mononuclear cells and TIL ex vivo, cells werestimulated for 5 hrs with PMA (0.2 μM) and Ionomycin (1 μg/ml), with BFA(10 μg/ml) present for the last 2½ hrs. Intracellular cytokine stainingwas performed using the CytoFix/CytoPerm kit from BD Bioscienceaccording to the manufacturer's instructions. Stained cells wereacquired on a LSRII and Fortessa flow cytometer, or the FACS AriaII (allBD), for cell sorting. Data were analyzed with FlowJo software(Treestar).

Cell sorting and T cell expansion: Cryopreserved PBMC and TIL werethawed and enriched for T lymphocytes using the T cell enrichment kitfrom Stemcell. For TIL enrichment, Epcam beads (StemCell) were added tothe cocktail. The enriched fractions were then labeled and populationsof interest were purified after cell sorting to 99% purity on a FACSAriaII (BD).

For TCR sequencing analysis, cell pellets were frozen until furtherprocessing.

For expansion of DN CD8+, SP CD8+ and DP CD8+ TIL as well as naïve andmemory CD8 from PBMC, T cells were sorted and cultured in completeRPMI-1640, supplemented with 2 mM glutamine, 1% (vol/vol) nonessentialamino acids, 1% (vol/vol) sodium pyruvate, penicillin (50 U/ml),streptomycin (50 ug/ml) and 10% fetal bovine serum (Hyclone). T cells(from 2000-5000 cells/well) were stimulated polyclonally with 1 μg/mlPHA (Sigma) in the presence of irradiated (4000 rad) allogeneic feedercells (2×10⁵ cells/well) and 10 ng/ml of rh IL-15 (Biolegend) in a 96well round-bottom plate (Corning/Costar). After 1 week, once T cellclusters formed, cells were split in an additional 96 well plate incomplete medium with IL-15, which was repeated again after 2 days,resulting in 4 identical replicates. The four replicates were thenpooled in a well of a 24 well plate at day 12. T cell lines weremaintained until analysis.

Microarray data acquisition: Samples for micro array were processed in asimilar manor to flow analysis. Tumor digest was completed in a 50 mlconical tube with a magnetic stir bar at room temperature for 1 hourwith Collagenase at 1 mg/ml (Sigma, C-5138), halyuronidase at 0.5 mg/ml(Sigma, H-6254) in RPMI (Life Technologies, 11875-093) with 0.3% humanalbumin (MP Biomedicals 823051) and 30 u/ml DNASE (Roche 04536282001).Following digest samples were filtered through a 70 μm filter. Sampleswere then diluted 1:2 with RPMI and layered onto Ficoll (GE, 17-1440-02)to enrich for lymphocytes through a centrifugation step. Enriched cellswere stained for CD3, CD8, CD103, CD39 and sorted using a BD FACSAriacell sorter. Sorted cells were lysed and RNA was purified usingDirect-zol RNA miniprep kit (zymo research) RNA was then reversetranscribed to cDNA and amplified. Amplified cDNA was hybridized on aAffymetrix Prime-View gene chip.

Microarray data analysis: CHP files, produced by the Affymetrix GeneChipCommand Console (AGCC) v. 3.1.1 and Affymetrix Expression Console v. 1.1software from the Affymetrix Primeview gene expression array were loadedusing the BRB ArrayTools, Version 4.5.1 (available on the internet,brb_nci.nih.gov/BRB-ArrayTools/) software, and annotated with theirsample type (i.e. DN, SP, DP). The Affymetrix Quality Control module wasrun to determine that the expression results were within specifications(applies the BioConductor R module: affy/affyQCReport). Differentialexpression between sample types was determined using the ClassComparison/Between Groups of Arrays BRBarray module, using a setsignificance threshold of univariate test (reports least-square andKolmogorov-Smirnov tests sorted by the p-value of the univariate test).The Graphics/Visualization of Samples module was then used to produce aMulti-Dimensional Analysis of the 3 groups using selected genes (appliesthe BRBarray R module: MDS.R).

In-vitro T cell activation: Naïve CD8 T cell subsets were isolated bymagnetic CD8 T cell enrichment (Stemcell), labeled with antibodiesagainst CD4, CD8, CD45RA and CCR7 and sorted. 1×10⁵ naïve T cells werecultured with anti-CD3/CD28 Dynabeads (Life Technologies) at a bead:Tcell ratio of 1:2 in the presence or absence of 2 ng/ml rh TGFβ-1 (R&D).After 24 hours, beads were removed by magnetic capture for half of theexperiment. Expression of activation and differentiation markers wasassessed at days 1, 2, 3, 4, 7 and 9.

Deep TCR sequencing of TCR VB gene and clonality analysis: Deepsequencing of the variable V-J or V-D-J regions of TCRβ genes wasperformed on genomic DNA of sorted T cell populations. DNA was extractedfrom circulating and tumor-resident CD8 T cell subsets ranging from1×10⁴-1×10⁵ cells (DNeasy Blood and Tissue Kit, Qiagen). The TCRβ CDR3regions were sequenced and mapped (ImmunoSEQ, Adaptive Biotech).Coverage per sample was >10×. Only data from productive rearrangementswere extracted from the ImmunoSEQ Analyzer platform for furtheranalysis. Clonality of the different T cell subsets was assessed bynucleotide sequence comparison of the 500 most abundant clones in eachsubset.

To compare the TCR Vβ overlap (or similarity) of two given populations,we used the Morisita's overlap index. The Morisita-Horn similarity indexaccounts for both, the number of common clonotypes and the distributionof clonotype sizes, and it is most sensitive to the clone sizes of thedominant clonotypes (ref Venturi et al., J Immunologi Meth, 2008).

Assessment of target cell recognition: 4-1BB and CD25 up-regulation andIFN-γ secretion: Upregulation of 4-1BB and CD25 as well as release ofIFN-γ were used as measures to assess recognition of tumor cells byexpanded autologous CD8 T cells. The coculture experiment was performed17-20 days after beginning the expansion. The day before, expanded Tcell were counted and starved overnight in medium without IL-15 todownregulate remaining expression of 4-1BB and CD25. Expanded CD8 Tcells (1×10⁵) were then cultured either alone or with tumor cells(autologous and allogeneic, ratio T cells:target cells=10:1). In someconditions, tumor cells were preincubated with 30 ug/ml of anti-MHCclass I blocking antibody (BD Bioscience, clone W6/32) for 3 h prior toadding T cells. As positive control, Nunc Maxisorp plates were coatedwith anti-CD3 antibody (OKT3) and T cells were added. All conditionswere plated in triplicate. After 24 hrs, supernatants were harvested andanalyzed by cytometric bead array (CBA) analysis. Cells were pooled foreach condition and labeled with a viability dye, followed by CD39,CD103, CD25 and 4-1BB cell surface staining. Cells were analyzed by flowcytometry. For the CBA, the manufacturer's protocol was followed. Inparticular, IFN-γ and TNF-α were analyzed.

Live target cell killing assay: T cell-mediated target cell killingassays were performed in the Incucyte Zoom System housed inside a cellincubator at 37° C./5% CO₂, based on the manufacturer's protocol (EssenBioscience). To assess T cell killing of autologous tumor cells,5000-10000 tumor cells (autologous and allogeneic tumor cell lines) wereseeded in triplicate into a 96-well flat-bottom plate to reach 10%confluence. Expanded autologous T cell subsets were counted and starvedo.n. without exogenous IL-15. 1×10⁵ T cells were then cultured with andwithout autologous and allogeneic tumor cells (ratio T cells:Tumorcells=10:1). In some conditions, anti-MHC class I antibody (BDBioscience, clone W6/32) was added. In all conditions, NucView 488Caspase 3/7 substrate (Essen Bioscience) was added to monitor activeCaspase 3/7. Plates were incubated for 24 hrs at 37° C. and four imageswere captured from three experimental replicates every hour using a 10×objective lens to visualize T cell killing and caspase 3/7 activity(green fluorescence). Green channel acquisition time was 400 ns. Forphase contrast, cell segmentation was achieved by applying a mask inorder to exclude the smaller T cells. An area filter was applied toexclude objects below 1000 μm². Green background noise was subtractedwith the Top-Hat method of background non-uniformity correction with aradius of 20 μm and a threshold of 2 green corrected units. Fluorescencesignal was quantified after applying the mask to the experiment. Amountof T cell killing/apoptosis was calculated by the Zoom software provided(Essen Bioscience).

Statistical analysis: Statistical tests were performed using Prismsoftware (GraphPad, San Diego Calif.). Significance was determined byone-way ANOVA analysis with Tukey correction, as noted in figurelegends.

Example 2 Results

Identification of tumor-reactive CD8 T cells is key to assess the leveland quality of the anti-tumor response in cancer patients and tounderstand the mode of action of new cancer treatment strategies such asimmunotherapy. Recently, human tumor-reactive CD8 T cells have beenidentified by the co-expression of CD103 and PD-1 in high grade serousovarin cancer (HGSC) and non-small cell lung cancer (NCLC). To furtherdefine their properties and function, tumor-infiltrating CD8 T cells(TIL) were sorted from two human ovarian tumors into CD103 positive andnegative subsets and their gene expression profile was determined bymicroarray. For this analysis, attention was focused on differentiallyexpressed cell surface molecules that could be easily detected by flowcytometry. One of the greatest differences observed in the gene arraycomparison that fulfilled the criteria was ENTPD1, a gene that encodesfor CD39 and is found on the cell surface (Table 1). To confirm the genearray result and get a better understanding of the diversity oftumor-infiltrating CD8 T cells, CD8 T cells isolated from a head andneck squamous cell carcinoma (HNSCC) patient were stained and gated onthe CD103+ population. CD39 and additional cell surface markersassociated with activation/exhaustion as well as developmental statewere examined. The complexity of the simultaneous analysis of all flowcytometry markers required a method called t-distributed stochasticneighbor embedding (t-SNE), widely used for mass cytometry data.Interestingly, high expression of CD39 on CD103+CD8 T cells overlappedwith high expression levels of PD-1 and CD69. Conversely, the CD103+CD8T cells expressed low levels of the IL-7R (CD127) (FIGS. 1A and 1B),suggesting a more effector-like T cell phenotype. Furthermore, thecombined expression of CD103 and CD39 on CD8 T cells resulted in theidentification of three distinct cell populations, which include theCD103−CD39− (double negative (DN) CD8), CD103+CD39− (single positive(SP) CD8) and CD103+CD39+ (double positive (DP) CD8). The DP CD8 T cellswere detected at relatively high frequencies in several human solidmalignancies including HNSCC, lung cancer, melanoma, ovarian cancer andrectal cancer (FIG. 1C), although melanoma had the highest frequency ofthese cells. In contrast, the DP CD8 T cells were present at quite lowfrequencies in a subset of HNSCC patients as well as most patients withcolon cancer and colorectal liver metastasis (CRLM) (FIGS. 1C and 1D).Surprisingly, TIL from one colon cancer patient contained a very highfrequency of DP CD8 T cells. This patient was diagnosed with Lynchsyndrome, a rare hereditary disorder caused by mutations in mismatchrepair genes leading to a higher rate of mutations. Interestingly,tumors with a greater frequency of mutations have a greater probabilityof having large number of neoantigens and those patients have a betteroutcome to checkpoint blockade immunotherapy as illustrated by prolongedprogression-free survival (Le D T, N Engl J Med 2015; Snyder A, N Engl JMed 2014; Van Allen E M, Science 2015; Rizvi N A, Science 2015).

It was then examined whether the DP CD8 T cells were found only at siteswhere tumor cells were present. To address this, several HNSCC patientswere analyzed where we obtained access to the primary tumor, metastaticlymph nodes (LN), uninvolved LNs and peripheral blood. In FIG. 1 eresults from a representative patient are shown, where the co-expressionof CD39 and CD103 by CD8 T cells was specifically found in the primarytumor and metastatic LN but absent or present at very low frequency onCD8 T cells in the peripheral blood and the uninvolved LN. Importantly,this expression profile was confirmed in the majority of HNSCC patientsthat were analyzed (FIG. 1F). Therefore, the results show that CD39expression on CD103+CD8 T cells identifies a population of CD8 T cellsspecifically induced within the tumor microenvironment.

To better understand the biology of DN, SP and DP CD8 T cells, the threecell populations were sorted directly ex vivo and their globalgene-expression profiles were determined by microarray. For thisanalysis, T cells were isolated from three HNSCC and two ovarian tumors.Comparison between DP and DN CD8 T cells identified 372 genesdifferentially expressed between these two cell populations. PrincipalComponent Analysis and unsupervised hierarchical clustering on thisselected gene list revealed that DP and DN CD8 T cells had distinct mRNAprofiles, whereas SP CD8 T cells displayed an intermediate genesignature (FIGS. 2 a and b ). Principal Component Analysis andunsupervised hierarchical clustering performed on this selected set ofgenes revealed that the gene expression profile was cell type specificand did not segregated by patient (FIG. 2A, 2B). When comparingtranscripts from the DP CD8 T cells relative to the DN CD8 T cells someof the greatest increases were associated with an activated/exhaustedphenotype such as MKI67, TNFRSF9, CTLA-4, HAVCR2, and GZMB. In contrast,the most down-regulated genes in DP CD8 T cells relative to DN CD8 Tcells were involved in T cell recirculation patterns, such as KLF2,CCR7, SELL, S1PR1, KLF2. This gene expression profile is reminiscent ofa T resident memory signature (FIG. 2C). Importantly, the expressionprofile for those activation and recirculation associated mRNAs wasconsistent across all five tumors (FIG. 2D). Therefore, the DP CD8 Tcells display a gene expression signature of cells undergoingantigen-driven stimulation and activation in the tumor, which also mayresult in the loss recirculation out of the tumor tissue.

To further assess the specific properties of tumor-infiltrating CD8 Tcells, the expression of differentiation and activation markers wasanalyzed at the protein level by flow cytometry. The DP CD8 T cellsexpressed higher levels of CD69 when compared to SP or DN CD8 T cells(FIG. 3A). CD69 is an activation molecule that is up-regulated after Tcell stimulation and antagonizes sphingosine 1-phosphate receptor 1(S1PR1)-mediated egress from tissues (Mackay 2015; Skon 2013). The DPCD8 T cells also exhibited lower levels of CCR7, IL7R (CD127) and CD28,indicative of an effector memory phenotype (ref). Interestingly, eventhough DN and SP CD8 T cells expressed PD-1, the DP CD8 T cellsexpressed significantly higher levels of this protein (FIG. 3B). Inaddition, CTLA-4, and TIM-3 were almost exclusively expressed within DPCD8 T cell population. Hence the DP CD8 T cells displayed a highlyactivated effector memory phenotype especially when compared to the DNand SP CD8 T cells. Moreover, the increased frequency of 4-1BB and Ki-67within the DP CD8 T cell population suggested that these cells wererecently activated and proliferating within the tumor, which isindicative of recent cognate antigen recognition.

The effector function of DN, SP and DP CD8 T cells was assessed directlyex vivo by analyzing their cytokine production at the single cell level.The DP CD8 T cells had a lower frequency of cells capable of producingIFN-γ and or TNF-α when compared to DN and SP CD8 T cells and similarresults were obtained in six HNSCC patients (FIG. 3C, 3D). In contrast,the DP CD8 T cells had an increased cytotoxic potential as demonstratedby a significantly higher frequency of granzyme B-positive cells (FIG.3E).

Given that DP CD8 T cells were only found at sites where tumor cellswere present, the factors responsible for this particular phenotype wereidentified to better understand the development of these cells. It isestablished that TGF-β drives expression of CD103 on T cells. TGF-β isproduced within the tumor microenvironment and plays a major role incancer progression through the induction of the epithelial-mesenchymaltransition leading to enhanced motility and invasion. CD39 expressionwas found on chronically stimulated/exhausted CD8 T cells in a mousemodel of chronic LCMV infection as well as in patients with chronic HCVand HIV infections (Gupta P K, PLOS Pathogen 2015), supporting a rolefor sustained TCR stimulation in its expression. Therefore, the kineticsof CD103 and CD39 upregulation were analyzed on naïve CD8 T cells afterTCR engagement in the presence or absence of TGF-β (FIG. 4 andsupplemental data). To address the role of sustained TCR stimulation inCD39 expression, T cell were stimulated continuously with CD3/CD28 beadsfor 9 days or the beads were removed 24 h after the initiation of theculture. CD39 expression was detected within 3 days of culture and itsexpression increased until day 9. CD103 was up-regulated when T cellswere stimulated in the presence of TGF-β with more than 80% of cellspositive after 7 days. Optimal CD103 and CD39 up-regulation was foundonly after sustained TCR stimulation. Importantly, the lack of CD39up-regulation in the absence of sustained TCR stimulation was not due tolimited T cell activation as the expression of other activation markerssuch as PD-1 was rapidly induced on the cells (FIG. 4 ). As opposed toCD103 expression, TGF-β had no effect on CD39 expression in the in vitroculture system. Thus, sustained TCR stimulation and TGF-β are thenecessary factors required to promote the expression of CD103 and CD39on CD8 T cells. Hence it appears that the DP CD8 T cells acquire theirphenotype upon repeated exposure to their cognate antigen leading tochronic TCR signaling within the tumor in a TGF-β-rich environment.

This data suggested that the DP CD8 T cells recognize their cognateantigens within the tumor site. If this is correct, it would lead toselective expansion of dominant TCR clonotypes, resulting in increasedclonality when compared to the other CD8 T cell populations. To addressthis, the TCRβ clonotypes were assessed within the DN, SP and DP CD8 Tcell populations as well as of total memory CD8 T cells from pairedblood and uninvolved LN by sequencing the highly variable CDR3 region ofthe TCRB genes. DP CD8 T cells were more oligoclonal as compared to anyother CD8 T cell population analyzed (FIG. 5A). The 30 most frequentclonotypes in each patient accounted for 56%, 61% and 66% of the DP CD8T cell population—but only 26% and 23% and 38% of the DN CD8 T cells andless than 20% of the memory peripheral CD8 T cells—in an HPV+ HNSCCtumor, an HPV− HNSCC tumor, and an ovarian tumor, respectively. The 30highest expanded DP CD8 T cell clonotypes were far less frequent in theDN CD8 T cell population, and represented less than 0.06% of the DN CD8T cell repertoire for these three patients. Interestingly, there werevery few shared TCR clonotypes between DP CD8 T cells and the othertumor-infiltrating CD8 T cell subsets (FIG. 5B). In contrast, a largernumber of the TCR clonotypes were shared between DN CD8 T cells and SPCD8 T cells. As predicted by the resident memory signature (FIG. 2D),the majority of TCR clonotypes present in DN CD8 T cells were alsoshared with memory CD8 T cells within the tumor uninvolved LN as well aswith memory CD8 T cells in the peripheral blood (R²=0.7710 and 0.2022,respectively), suggesting that the DN CD8 T cells were able torecirculate, potentially sensing the tumor environment withoutultimately recognizing their cognate antigen (FIG. 5C). In comparison,very few clonotypes present within DP CD8 T cells were detected in theuninvolved LN or peripheral blood. Calculation of the Morisita index, anabundance-based similarity index that determines the overlap between twopopulations further supported our results and showed consistency of thisfinding across 5 patient samples (FIG. 5D). Collectively, these resultssuggest that the DP CD8 T cells encounter their cognate antigen withinthe tumor site, which results in their activation and local expansion oftumor-specific T cell clones.

If this is correct, then the DP CD8 TIL should be enriched for tumorreactivity. Hence autologous tumor cell lines were generated from fourmelanoma tumors and two HNSCC tumors and we determined whether the DPCD8 TIL were enriched for tumor reactivity and killing. CD8 T cells weresorted directly from tumor digests based on the expression of CD103 andCD39 expression and the three CD8 T cell populations were expanded invitro. Following expansion the DN, SP and DP CD8 TIL were screened forreactivity against autologous tumor cell lines as assessed by 4-1BBup-regulation as well as IFN-γ secretion. In all six patients tested itwas found that the DP CD8 T cells were highly enriched for tumorreactivity when compared to the DN and SP CD8 TIL (FIG. 6A). Theenrichment in tumor-reactive CD8 TIL among DP CD8 cells was illustratedby up to 87% reactive T cells at the highest tumor to T cell ratio forpatient 1. Recognition of autologous tumor was specific and MHC class Irestricted as no reactivity in the DP TIL subset was detected followingMHC class I blockade or when cells were cultured with an allogeneictumor cell line (FIG. 6B). To address whether the tumor-reactive DP CD8T cells were capable of killing autologous tumor cells, the expanded Tcell subsets were cocultured with autologous tumor cells and monitoredfor tumor-specific killing using the Incucyte live-cell analysis system.This system allows for visualization of caspase 3/7-dependent apoptosisby microscopy at 37° C. in real time. In accord with 4-1BB upregulation,only the DP CD8 T cells killed autologous tumor cells as illustrated bythe increasing number of caspase 3/7+ events (FIG. 6C, 6D). This killingwas MHC-class I dependent as the MHC-class I antibody W6/32 blocked thiseffect. In contrast there was little to no autologous tumor cell killingobserved by the DN CD8 T cells.

Finally, the relationship between the frequency of DP CD8 TIL andpatient survival was assessed in surgical samples. This analysis focusedon a small cohort of HNSCC patients (n=62). Following surgery, thefrequency of DP CD8 T cells among total CD8 TIL was determined withinprimary tumors by flow cytometry and patients then received standard ofcare treatment(s). Patients were segregated based on the frequency of DPCD8 T cells, with a high and a low group relative to the averagefrequency of DP CD8 T cells for this cohort. Using this strategy, it wasfound that patients whose tumors had a higher percentage of DP CD8 TILat the time of surgery correlated with increased overall survival (OS)(FIG. 6E), which showed greater significance in the HPV− negativesubgroup (FIG. 6F). Collectively, it was shown that CD103 and CD39co-expression strongly enriches for tumor-reactive CD8 TIL and theirfrequency correlated with an increase in overall survival in HNSCCpatients.

Based on these results, it was addressed whether the TCR clonotypespresent at high frequencies in DP CD8 TIL were indeed tumor-specific. DPCD8 TIL were cocultured with autologous tumor cells for 20 hrs, followedby sorting of the 4-1BB+CD25+CD8 T cells. The majority of the clonotypespresent at high frequency ex-vivo were found within the 4-1BB+CD25+fraction of DP CD8 TIL when these T cells were co-cultured withautologous tumor lines, demonstrating that they were tumor reactive(FIGS. 7A. 7B). The data imply that utilizing TCRs from the DP CD8population, would be therapeutic in an adoptive transfer setting. Ofnote, a strong overlap of the DP CD8 TIL TCR repertoire was observedbetween the primary tumor and metastatic LN within the same patient, intwo different HNSCC samples (FIG. 7C). This result is significant as itsuggests that the same DP CD8 TIL TCR could be isolated from either ametastatic LN or primary tumor and used for TCR therapy.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method of isolating a nucleic acid encoding a T cellreceptor (TCR) that specifically binds a tumor cell antigen, comprising:isolating CD39+CD103+CD8+ T cells from a sample from a subject with atumor expressing the tumor cell antigen, and cloning a nucleic acidmolecule encoding a TCR from the CD8⁺CD39⁺CD103⁺ T cells, therebyisolating the nucleic acid molecule encoding the TCR.
 2. The method ofclaim 1, wherein the tumor is a head and neck squamous cell carcinoma,lung cancer, melanoma, ovarian cancer renal cell carcinoma, bladdercancer, cervical cancer, liver cancer, prostate cancer, breast cancer,glioblastoma or rectal cancer.
 3. The method of claim 1, wherein thesample is peripheral blood or a tumor biopsy.
 4. The method of claim 1,further comprising expanding the CD8⁺CD39⁺CD103⁺ T cells in vitro priorto cloning the T cell receptor.
 5. The method of claim 1, wherein thesubject is human.
 6. An isolated nucleic acid molecule encoding a TCRproduced by the method of claim
 1. 7. An isolated TCR encoded by anucleic acid molecule encoding a TCR produced by the method of claim 1.8. An isolated host T cell transfected with the nucleic acid molecule ofclaim
 6. 9. The host T cell of claim 8, wherein the TCR is autologous tothe subject.
 10. The isolated host T cell of claim 8, wherein the TCR isallogeneic to the subject.
 11. A method of treating a subject with atumor, comprising: cloning a nucleic acid molecule encoding a TCR fromCD8⁺CD39⁺CD103⁺ T cells from a patient with a tumor; transfecting hostcells with the TCR, and administering a therapeutically effective amountof the host cells to the subject, thereby treating the tumor in thesubject.
 12. The method of claim 11, wherein the tumor is a solid tumor.13. The method of claim 12, wherein the tumor is a head and necksquamous cell carcinoma, lung cancer, melanoma, ovarian cancer renalcell carcinoma, bladder cancer, cervical cancer, liver cancer, prostatecancer, breast cancer, glioblastoma or rectal cancer.
 14. The method ofclaim 11, wherein the subject is a human.
 15. The method of claim 11,wherein the host cells are T cells or natural killer cells.
 16. Themethod of claim 15, wherein the host cells are the T cells, and the Tcells are CD3+ T cells.
 17. The method of claim 11, wherein the hostcells are autologous to the subject.
 18. The method of claim 11, whereinthe subject and the patient are the same individual.
 19. The method ofclaim 11, further comprising expanding the CD8⁺CD39⁺CD103⁺ T cells invitro prior to cloning the T cell receptor.
 20. A method of treating asubject with a tumor, comprising: administering a therapeuticallyeffective amount of the host cells comprising a nucleic acid moleculeencoding a TCR isolated from CD8⁺CD39⁺CD103⁺ T cells to the subject,thereby treating the tumor in the subject.